Vaccine comprising amb a 1 peptides for use in the treatment of ragweed allergy

ABSTRACT

The present invention relates to compositions for preventing or treating allergy to ragweed by tolerisation. The compositions are based on combinations of peptide fragments derived from the major allergen in ragweed pollen, Amb a 1. The invention also relates to products, vectors and formulations which may be used to provide polypeptides of the invention in combination. The invention further relates to in vitro methods for determining whether T cells recognize a polypeptide of the invention, and for determining whether an individual has or is at risk of a condition characterized by allergic symptoms in response to a ragweed allergen.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/917,756 filed Jun. 14, 2013, which is a divisional of U.S. patentapplication Ser. No. 13/057,386 filed May 27, 2011, which is a 35 U.S.C.371 national stage filing of International Application No.PCT/GB2009/001986 filed Aug. 14, 2009, which claims priority to GBApplication No. 0814986.6 filed Aug. 15, 2008; PCT application No.PCT/GB08/002781 filed Aug. 15, 2008; PCT application No. PCT/GB08/002779filed Aug. 15, 2008; GB Application No. 0815218.3 filed Aug. 20, 2008and EP Application No. 09251252.4 filed May 1, 2009. The contents of theaforementioned applications are hereby incorporated by reference.

INCORPORATING BY REFERENCE

The contents of the following priority applications are incorporatedherein by reference: United Kingdom Patent Application No. 0814986.6filed 15 Aug. 2008; International Patent Application No.'sPCT/GB08/002781 and PCT/GB08/002779, both filed 15 Aug. 2008; UnitedKingdom Patent Application No. 0815218.3 filed 20 Aug. 2008 and EuropeanPatent Application No. 09251252.4 filed 1 May 2009.

FIELD OF THE INVENTION

The present invention relates to compositions for preventing or treatingallergy to ragweed.

BACKGROUND OF THE INVENTION

T-cell antigen recognition requires antigen presenting cells (APCs) topresent antigen fragments (peptides) on their cell surface inassociation with molecules of the major histocompatibility complex(MHC). T cells use their antigen specific T-cell receptors (TCRs) torecognise the antigen fragments presented by the APC. Such recognitionacts as a trigger to the immune system to generate a range of responsesto eradicate the antigen which has been recognised.

Recognition of external antigens by the immune system of an organism,such as man, can in some cases result in diseases, known as atopicconditions. Examples of the latter are the allergic diseases includingasthma, atopic dermatitis and allergic rhinitis. In this group ofdiseases, B lymphocytes generate antibodies of the IgE class (in humans)which bind externally derived antigens, which are referred to in thiscontext as allergens since these molecules elicit an allergic response.Production of allergen-specific IgE is dependent upon T lymphocyteswhich are also activated by (are specific for) the allergen.Allergen-specific IgE antibodies bind to the surface of cells such asbasophils and mast cells by virtue of the expression by these cells ofsurface receptors for IgE.

Crosslinking of surface bound IgE molecules by allergen results indegranulation of these effector cells causing release of inflammatorymediators such as histamine, 5-hydroxtryptamine and lipid mediators suchas the sulphidoleukotrienes. In addition to IgE-dependent events,certain allergic diseases such as asthma are characterised byIgE-independent events.

Allergic IgE-mediated diseases are currently treated with agents whichprovide symptomatic relief or prevention. Examples of such agents areanti-histamines, β2 agonists, and glucocorticosteroids. In addition,some IgE-mediated diseases are treated by desensitisation proceduresthat involve the periodic injection of allergen components or extracts.Desensitisation treatments may induce an IgG response that competes withIgE for allergen, or they may induce specific suppressor T cells thatblock the synthesis of IgE directed against allergen. This form oftreatment is not always effective and poses the risk of provokingserious side effects, particularly general anaphylactic shock. This canbe fatal unless recognised immediately and treated with adrenaline. Atherapeutic treatment that would decrease or eliminate the unwantedallergic-immune response to a particular allergen, without altering theimmune reactivity to other foreign antigens or triggering an allergicresponse itself would be of great benefit to allergic individuals.

Ragweed allergens are universally recognised as a major cause ofallergic diseases in humans and animals, including asthma, allergicrhinitis and allergic dermatitis. Proteins present in ragweed pollen areparticularly important. For example, approximately 75% of hayfeversufferers in the United States are allergic to ragweed pollen. Hayfeveris the common term for a form of seasonal allergy characterised bysneezing, runny nose and itching eyes. The term “hayfever” arose becausethis form of allergic disease is most prevalent during “haying season”,which corresponds to the flowering season of many plants, that is whenthey release the highest quantities of pollen. It is particularlyprevalent from late summer to early Autumn, typically from the end ofJune to the end of September (in the Northern Hemisphere).

It has been calculated that for adults in the United States, hayfever isthe 5th leading chronic disease and a major cause of work absenteeism,resulting in nearly 4 million missed or lost workdays each year,resulting in a total cost of more than $700 million in total lostproductivity. Allergies are also the most frequently reported chroniccondition in children, limiting activities for more than 40% of them.Each year, allergies account for more than 17 million outpatient officevisits in the United States; seasonal allergies such as hayfever accountfor more than half of these allergy visits.

A therapeutic or preventative treatment would therefore be of greatbenefit to humans that suffer or are at risk of suffering from ragweedallergy.

SUMMARY OF THE INVENTION

Ragweed allergy is typically caused by Common ragweed (Ambrosiaartemisiifolia). The major allergen in ragweed pollen is Amb a 1. Thisprotein exists as a number of different isoforms, Amb a 1.1, 1.2, 1.3and 1.4. The present inventors have discovered that certain combinationsof peptide fragments derived from the Amb a 1 proteins are particularlyuseful in desensitising individuals to these allergens. The polypeptidecombinations of the invention have been selected for their ability bindto many MHC Class II molecules, be highly soluble, to not triggerhistamine release from basophils drawn from a panel of ragweed allergicindividuals and to induce a cytokine response in a high proportion ofsubjects from a panel of ragweed allergic individuals. The compositions,products, vectors and formulations of the invention may therefore beprovided to individuals for preventing or treating allergy to ragweed bytolerisation.

The peptides of the invention were selected as potential T cell epitopesthrough in silico methods. When regions containing epitopes wereidentified, they were further analysed to determine which of them werehighly conserved between the four different Amb a 1 isoforms. Thesecandidate polypeptides were then further screened for potential use intolerisation. More specifically, they were analysed for solubilitycharacteristics, and the ability to induce cytokine release from PBMCderived from ragweed allergic individuals. In some instances, thepeptide sequences were engineered to improve solubility and/or reducedimer formation. A difficulty associated with approaches todesensitisation based on peptide immunisation lies in how to select anappropriate size and region of the allergen as the basis for the peptideto be used for immunisation. The size of the peptide of choice iscrucial. If the peptide is too small, the vaccine would not be effectivein inducing an immunological response. If the peptides are too large, orif the whole antigen is introduced into an individual, there is the riskof inducing adverse reactions, such as anaphylaxis, which may be fatal.This risk may be greater if peptides are poorly soluble.

The polypeptides of the invention have been selected to retain T cellspecificity whilst being small enough in size to not possess significanttertiary structure that would enable them to retain the conformation ofan IgE-binding epitope of the whole molecule. The polypeptides of theinvention therefore do not induce significant crosslinking of adjacentspecific IgE molecules on cells such as mast cells and basophils andhave been shown not to cause significant histamine release from humanbasophils.

An advantage of the invention is the ability of the peptides to broadlytarget Major Histocompatibility Complex (MHC) molecules. T cellreceptors (TCRs) are highly variable in their specificity. Variabilityis generated, as with antibody molecules, through gene recombinationevents within the cell. TCRs recognise antigen in the form of shortpeptides bound to molecules encoded by the genes of the MajorHistocompatibility Complex (MHC). These gene products are the samemolecules that give rise to “tissue types” used in transplantation andare also referred to as Human Leukocyte Antigen molecules (HLAs) whichterms may be used interchangeably. Individual MHC molecules possesspeptide binding grooves which, due to their shape and charge are onlycapable of binding a limited group of peptides. The peptides bound byone MHC molecule may not necessarily be bound by other MHC molecules.

When a protein molecule such as an antigen or allergen is taken up byantigen presenting cells such as B lymphocytes, dendritic cells,monocytes and macrophages, the molecule is enzymatically degraded withinthe cell. The process of degradation gives rise to peptide fragments ofthe molecule which, if they are of the appropriate size, charge andshape, may then bind within the peptide binding groove of certain MHCmolecules and be subsequently displayed upon the surface of antigenpresenting cells. If the peptide/MHC complexes are present upon theantigen presenting cell surface in sufficient numbers they may thenactivate T cells which bear the appropriate peptide/MHC-specific T cellreceptors. Due to the polymorphic nature of the MHC, individuals in anoutbred population such as man will express different combinations ofMHC molecules on their cell surfaces. Since different MHC molecules canbind different peptides from the same molecule based on the size, chargeand shape of the peptide, different individuals will display a differentrepertoire of peptides bound to their MHC molecules. Identification ofuniversal MHC-binding peptide epitopes in an outbred population such asman is more difficult than in inbred animals (such as certain strains oflaboratory mice). On the basis of differential MHC expression betweenindividuals and the inherent differences in peptide binding andpresentation which this brings, it is unlikely that a single peptide canbe identified which will be of use for desensitisation therapy in man.

Another advantage of the invention is the selection of peptides andpeptide combinations on the basis of responses observed in PBMCs freshlyisolated from ragweed allergic individuals. In contrast to artefactualscenarios where clonal cell lines are established from allergic patientsin order to test T cell responses, the evaluation of ex vivo responsesof freshly isolated PBMCs allows for a representative view of therelative population importance of different peptides without potentialdistortion induced by the culture process.

The peptide combinations of the invention, however, provide a broadcoverage of efficacy over the human population by targeting multipledifferent MHC molecules. This broad coverage is illustrated by theability of peptide combinations of the invention to cause a positivecytokine response in many individuals within the population. A vaccineformulated with the peptides of the invention would therefore have broadutility.

The inventors' work has produced peptide combinations with the followingcharacteristics:

-   -   the combination induces a cytokine response in a high proportion        of subjects from a panel of ragweed allergic individuals    -   the peptides of the combinations are soluble    -   the peptides of the combinations do not induce significant        histamine release in a panel of ragweed allergic individuals.

Accordingly, the present invention provides a composition for use inpreventing or treating allergy to ragweed by tolerisation comprising atleast one polypeptide selected from SEQ ID Nos. 1 to 31. Typically, thecomposition comprises at least four polypeptides, wherein thepolypeptides are independently selected from any of the following:

(i) a polypeptide of SEQ ID NO's 1 to 31; or

(ii) a variant of a polypeptide according to (i), wherein said variantis a polypeptide of length 9 to 30 amino acids that comprises a regionconsisting of:

-   -   any of the sequences of (i); or    -   a sequence which has at least 65% homology to any of the        sequences of (i) which sequence is capable of tolerising an        individual to any of the sequences of (i); or

(iii) a variant of a polypeptide according to (i), wherein said variantis a polypeptide of length 9 to 30 amino acids that comprises a regionconsisting of a sequence that represents either:

-   -   a fragment of any of the sequences of (i); or    -   a homologue of a fragment of any of the sequences of (i),        which sequence is capable of tolerising an individual to any of        the sequences of (i) and has a length of at least 9 amino acids,        and wherein said homologue has at least 65% homology to any 9        contiguous amino acids in any of the sequences of (i).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 show the proportion of individuals responsive to differentpeptide combinations of the invention measured by production of IL-13 orIFN-gamma.

FIG. 2 shows the average production of IL-10 (pg/ml) by PBMCs fromragweed allergic individuals when stimulated with different peptides ofthe invention.

FIG. 3 shows the level of IL-10 (pg/ml) produced by PBMCs from ragweedallergic individuals when stimulated with different peptidecombinations.

DESCRIPTION OF THE SEQUENCES MENTIONED HEREIN

SEQ ID NOS: 1 to 31 provide the polypeptide sequences of the invention.SEQ ID NOS: 32 onwards provide additional sequences.

DETAILED DESCRIPTION OF THE INVENTION

The invention concerns peptides and combinations of peptides which canbe used in tolerisation. Such peptides may comprise, consist of, orconsist essentially of the sequences shown in any of SEQ ID NO's. 1 to31). Variants of these specific peptides may also be used. The variantsmay comprise, consist of, or consist essentially of sequences which arefragments of either any of SEQ ID NO's 1 to 31 or homologues of any ofSEQ ID NO's 1 to 31.

In one embodiment the invention relates to a composition for use inpreventing or treating allergy to ragweed. The composition typicallycomprises or consists at least four, five, six, seven, eight, nine, ten,eleven, or twelve polypeptides, up to a maximum of thirteen. In otherwords, the composition comprises between four and thirteen polypeptides.The polypeptides are independently selected from any of the following:

(i) a polypeptide of SEQ ID NO's 1 to 31; or

(ii) a variant of a polypeptide according to (i), wherein said variantis a polypeptide of length 9 to 30 amino acids that comprises a regionconsisting of:

-   -   any of the sequences of (i); or    -   a sequence which has at least 65% homology to any of the        sequences of (i) which sequence is capable of tolerising an        individual to any of the sequences of (i), or

(iii) a variant of a polypeptide according to (i), wherein said variantis a polypeptide of length 9 to 30 amino acids that comprises a regionconsisting of a sequence that represents either:

-   -   a fragment of any of the sequences of (i); or    -   a homologue of a fragment of any of the sequences of (i),        which sequence is capable of tolerising an individual to any of        the sequences of (i) and has a length of at least 9 amino acids,        and wherein said homologue has at least 65% homology to any 9        contiguous amino acids in any of the sequences of (i).

The invention also provides products and formulations comprising thepolypeptides of the invention and compositions, products and vectorscomprising polynucleotides capable of expressing the polypeptides of theinvention for use in preventing or treating ragweed allergy bytolerisation. Such tolerisation will typically be to an epitope (forexample a MHC class II epitope) present in any of SEQ ID NO's 1 to 31.

Ragweed Species

The ragweed species Ambrosia artemisiifolia (common ragweed), and to alesser extent Ambrosia trifida (giant ragweed), are responsible for ahigh proportion of ragweed allergy worldwide, particularly allergiesassociated with ragweed pollen, such as hayfever. Common ragweed isnative to North America, but has spread to most continents worldwide.Ragweeds bloom in the northern hemisphere from early July-mid August oruntil cooler weather arrives. Ragweed is a pioneer plant which is welladapted to colonising newly disturbed ground. While in natural habitatsit is often restricted by competition with other plants, but in areaswhere humans have cleared existing vegetation, ragweed quickly becomeswidely and aggressively established. Thus, ragweed is very abundantalong rural roadsides, fence lines, waste lands, new excavations,cultivated fields, gardens, and poorly kept lawns. As such it iswell-adapted to wide variety of climates, can tolerate a wide soil pHrange (from about 4.5 to about 8.5), and is also resistant to highsalinity.

Peptide Fragments of Ragweed Pollen Allergens

The major allergen in ragweed pollen is Amb a 1. This protein exists asa number of different isoforms, Amb a 1.1, 1.2. 1.3 and 1.4. Theseisoforms are set out in full in Example 1. The present inventors haveidentified the regions in Amb a 1 which comprise MHC Class II-binding Tcell epitopes and which are highly conserved between isoforms (seeanalysis in Example 1). Based on this information, peptides derived fromthe relevant regions of Amb a 1 are suitable for preventing or treatingragweed allergy by tolerisation to all isoforms of Amb a 1.

The terms “peptide” and “polypeptide” are used interchangeably herein.The above proteins are also referred to herein as “the allergens”.

Tables 3, 4 and 6 set out the sequences of the peptides of theinvention, where appropriate indicating the parent protein from whicheach peptide derives.

Peptide Combinations

The composition typically comprises a combination of at least threedifferent polypeptides of the invention, up to a maximum of thirteendifferent polypeptides. Accordingly, the composition of the inventionmay consist of three, four, five, six, seven, eight, nine, ten, eleven,twelve or thirteen peptides.

The polypeptide combinations in the composition of the invention areselected to provide as broad a coverage of the human population aspossible, i.e. the composition of the invention will produce an immuneresponse in a high proportion of ragweed allergic individuals,preferably more than 30%, 40%, 45%, 50%, 60% or 70% of ragweed allergicindividuals in a panel or population of such individuals. The number ofindividuals in a population of ragweed allergic individuals may be anysuitable number, typically at least 20, 30, 40, 50, 60, 70, 80, or atleast 100 individuals. Preferably the population has MHC allelefrequencies within the range of frequencies that are representative ofthe Caucasian population. Reference population allele frequencies for 11common DRB 1 allele families are shown in

Table 1 (Data from HLA Facts Book, Parham and Barber).

The composition of the invention typically comprises:

-   -   at least one polypeptide selected from a polypeptide of RGW01,        RGW01A or RGW01B, or a variant thereof; and    -   at least one polypeptide selected from a polypeptide of RGW03,        RGW03A or RGW03B, or a variant thereof; and    -   at least one polypeptide selected from a polypeptide of RGW04 or        RGW04A, or a variant thereof.

Optionally, the composition may additionally comprise at least oneadditional polypeptide selected from a polypeptide of any of RGW02,RGW09, RGW06 or RGW06A, RGW10, RGW10A, RGW05 or RGW05A, or a variantthereof.

Optionally, the composition may additionally comprise at least oneadditional polypeptide selected from a polypeptide of any of RGW07,RGW07C, RGW07D, or a variant thereof. The at least one additionalpolypeptide is preferably a polypeptide of RGW07D, or a variant thereof.

More specifically, in one embodiment, the invention therefore provides acomposition comprising between three and thirteen polypeptides,consisting of:

-   -   a) at least one of the polypeptides of RGW01, RGW01A or RGW01B,        or a variant thereof, preferably RGW01; and    -   b) at least one of the polypeptides of RGW03, RGW03A or RGW03B,        or a variant thereof, preferably RGW03B; and    -   c) at least one of the polypeptides of any of RGW04 or RGW04A,        or a variant thereof, preferably RGW04A; and optionally    -   d) at least one, preferably two, most preferably three, of the        polypeptides of any of RGW02, RGW09, RGW06, RGW06A, RGW10,        RGW10A, RGW05 or RGW05A, or a variant thereof, preferably RGW02        and/or RGW06A and/or RGW05; and optionally    -   e) at least one of the polypeptides of any of RGW07, RGW07C,        RGW07D, or a variant thereof, preferably RGW07D.

In other words, one specific embodiment of the invention provides acomposition for use in the prevention or treatment of ragweed allergy bytolerisation comprising between three and thirteen peptide sequences,wherein the composition consists of:

-   -   a) at least one of the polypeptides with the following        sequences:

RGW01 (SEQ ID NO: 1) GMIKSNDGPPI; RGW01A (SEQ ID NO: 2) GLIKSHDGPPV;RGW01B (SEQ ID NO: 3) GLIKSNDGPAA;

or a variant thereof, and;

-   -   b) at least one of the polypeptides with the following        sequences:

RGW03 (SEQ ID NO: 7) KDLLENGAIFVTSG; RGW03A (SEQ ID NO: 8)DVFENGAIFVPSG; RGW03B (SEQ ID NO: 9) RDLLENGAIFLPSG;

or variants thereof and;

-   -   c) at least one of the polypeptides with the following        sequences:

RGW04 (SEQ ID NO: 10) KAGMIPAEPGEA; RGW4A (SEQ ID NO: 11) SAGMIPAEPGEA;

or variants thereof and optionally;

-   -   d) at least one of the polypeptides with the following        sequences:

RGW02 (SEQ ID NO: 4) GSSQIWIDHSSLSKS; RGW04 (SEQ ID NO: 10)KAGMIPAEPGEA; RGW4A (SEQ ID NO: 11) SAGMIPAEPGEA; RGW06 (SEQ ID NO: 14)VVNSDKTIDGRGVKVE; RGW06A (SEQ ID NO: 15) AINNDKTIDGRGAKVE; RGW09(SEQ ID NO: 26) ETRRSLKTSGAYN; RGW10 (SEQ ID NO: 27) FGFFQVVNNNYD;RGW10A (SEQ ID NO: 28) HGFFQVVNNNYD; RGW05 (SEQ ID NO: 12)KEGTLRFAAAQNRP; RGW05A (SEQ ID NO: 13) KEGTLRFGAAQNRP;

or variants thereof and optionally;

-   -   e) at least one of the polypeptides with the following        sequences:

RGW07 (SEQ ID NO: 16) GEAAIKLTSSAGVLS; RGW07C (SEQ ID NO: 19)KGEAAIKLTSSAGVLSK; RGW07D (SEQ ID NO: 20) KGEAAIKLTSSAGVLSKK;

or variants thereof.

It will be appreciated that (a) to (e) above represent stringent andhighly selective criteria for the identification of suitablecombinations of the invention. For example, if one were to select sixpeptides at random from the sequences of the invention there would benearly a million possible combinations to choose from. By contrast, itis useful to consider an example of a combination of six polypeptides inwhich the above criteria are applied. For example, consider acombination wherein the following polypeptides are selected:

i) any one of the polypeptides of RGW01, RGW01A or RGW01B, and any oneof the polypeptides of RGW03, RGW03A or RGW03B and any one of thepolypeptides of RGW04 and RGW04A; and

ii) three further polypeptides selected from the polypeptides of any ofRGW02, RGW09, RGW06, RGW06A, RGW10, RGW10A, RGW05 or RGW05A; and finally

Based on such a selection, the number of possible combinationsrepresents a minute fraction of the total available combinations if thecriteria determined by the inventors are not applied.

On the basis of the above, a particularly preferred combination of theinvention comprises or consists of the polypeptides of RGW01, RGW03B,RGW04A, RGW02, RGW05 and RGW06A, or variants thereof.

A further preferred combination comprises of consists of thepolypeptides of RGW01, RGW03B, RGW04A, RGW02, RGW05, RGW06A and RGW07D.

Subject to the above, the composition may optionally comprise furtherpolypeptides up to a total of thirteen unique polypeptides. Thesefurther polypeptides relate to (i.e. are typically homologues and/orfragments of) the other sequences, i.e. SEQ ID NOS: 1 to 31, that arenot amongst the polypeptides already selected. The further peptides aretypically functional variants of one of the peptides of SEQ ID NO's 1 to31. The further polypeptides may be identical to any of SEQ ID NOS: 1 to31. The composition may therefore comprise up to thirteen differentpolypeptides as provided in any of SEQ ID NO: 1 to 31. However, theoptional further polypeptides do not need to be 100% identical to any ofSEQ ID NO: 1 to 31. They are preferably at least 65% identical to atleast 9 (for example at least 10, 11, 12 or 13) or more contiguous aminoacids in any of SEQ ID NO: 1 to 31, not already selected amongst thepreviously selected polypeptide(s). These contiguous amino acids maycomprise a MHC class II epitope, for example which binds to any of theMHC molecules mentioned herein. In other words, the composition mayoptionally comprise further polypeptides up to a total of thirteenunique polypeptides, wherein the further polypeptides:

-   (i)comprise a sequence having at least 65% sequence identity to at    least 9 or more contiguous amino acids in any of SEQ ID NO: 1 to 31    above not selected in (a) to (e) above; and-   (ii) are 9 to 30 amino acids in length.    wherein each different polypeptide is for simultaneous, separate or    sequential use in the prevention or treatment of ragweed allergy by    tolerisation.

In more detail therefore, the invention provides a product containingbetween three and thirteen polypeptides as defined in (a) to (e) above;and optionally:

-   -   (f) A polypeptide:        -   (i) comprising sequence having at least 65% sequence            identity to at least 9 or more contiguous amino acids in any            of SEQ ID NO: 1 to 31 not selected in a), to d) above; and        -   (ii) 9 to 30 amino acids in length; and optionally    -   (g) A polypeptide as defined in f), but additionally not        selected in f) above; and optionally    -   (h) A polypeptide as defined in f), but additionally not        selected in f) to g) above; and optionally    -   (i) A polypeptide as defined in f), but additionally not        selected in f) to h) above; and optionally    -   (j) A polypeptide as defined in f), but additionally not        selected in f) to i) above; and optionally    -   (k) A polypeptide as defined in f), but additionally not        selected in f) to j) above) above; and optionally    -   (l) A polypeptide as defined in f), but additionally not        selected in f) to k) above; and optionally    -   (m) A polypeptide as defined in f), but additionally not        selected in f) to l) above; and optionally    -   (n) A polypeptide as defined in f), but additionally not        selected in f) to m) above; and optionally    -   (o) A polypeptide as defined in f), but additionally not        selected in f) to n) above; and optionally (p) A polypeptide as        defined in f), but additionally not selected in f) to o) above        for simultaneous, separate or sequential use in the prevention        or treatment of ragweed allergy by tolerisation.

Another embodiment of the invention is a composition for use inpreventing or treating allergy to ragweed by tolerisation comprising oneor more polypeptide, wherein the polypeptide is selected from any of thefollowing:

(i) a polypeptide of any of SEQ ID NO's. 1 to 31;or

(ii) a variant of a polypeptide according to (i), wherein said variantis a polypeptide of length 9 to 30 amino acids that comprises a regionconsisting of:

-   -   any of the sequences of (i); or    -   a sequence which has at least 65% homology to any of the        sequences of (i) which sequence is capable of tolerising an        individual to any of the sequences of (i), or

(iii) a variant of a polypeptide according to (i), wherein said variantis a polypeptide of length 9 to 30 amino acids that comprises a regionconsisting of a sequence that represents either:

-   -   a fragment of any of the sequences of (i); or    -   a homologue of a fragment of any of the sequences of (i),        which sequence is capable of tolerising an individual to any of        the sequences of (i) and has a length of at least 9 amino acids,        and wherein said homologue has at least 65% homology to any 9        contiguous amino acids in any of the sequences of (i).

The compositions or products of the invention may comprise variants ofany of sequences defined above. The variant typically comprises 1, 2, 3or more of the MHC class II epitopes present in the correspondingpeptide of SEQ ID NO: 1 to 31.

Functional variants are mentioned herein. Such variants may be able totolerise an individual to a class II MHC epitope present in thecorresponding peptide of SEQ ID NO: 1 to 31, and thus it will typicallycomprise sequence that binds to the same MHC class II molecule and/or isrecognised by a T cell which recognises the corresponding epitope in thepolypeptide of SEQ ID NO: 1 to 31.

Variants of SEQ ID NO's 1 to 31 may be fragments derived by truncation.Truncation refers to the removal of one, two, three, four, five, six,seven, eight, nine, ten or more amino acids from the N and/or C-terminalends of a polypeptide of SEQ ID NOS. 1 to 31.

Fragments may also be generated by one or more internal deletions,provided that the core 9 amino acids that makes up the T cell epitope isnot substantially disrupted.

For example, a variant of SEQ ID NO: 1 may comprise a fragment of SEQ IDNO: 1, i.e. a shorter sequence. This may include a deletion of one, two,three, four, five, six, seven, eight, nine, ten or more amino acids fromthe N-terminal end of SEQ ID NO: 1 or from the C-terminal end of SEQ IDNO: 1. Such deletions may be made from both ends of SEQ ID NO: 1. Avariant of SEQ ID NO: 1 may include additional amino acids (for examplefrom the sequence of the parent protein from which the peptide derives)extending beyond the end(s) of SEQ ID NO: 1. A variant may include acombination of the deletions and additions discussed above. For example,amino acids may be deleted from one end of SEQ ID NO: 1, but additionalamino acids from the full length parent protein sequence may be added atthe other end of SEQ ID NO: 1. The same discussion of variants abovealso applies to SEQ ID NOS: 2 to 31. A preferred variant of SEQ ID NO:20 is the peptide KKGEAAIKLTSSAGVLSK (SEQ ID NO: 150).

A variant peptide may include one or more amino acid substitutions fromthe amino acid sequence of any of SEQ ID NOS: 1 to 31 or a fragmentthereof. A variant peptide may comprise sequence having at least 65%sequence identity to at least 9 or more contiguous amino acids in any ofSEQ ID NOS: 1 to 31. More preferably a suitable variant may comprise atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 98% amino acid identity to at least 9 contiguousamino acids of any of SEQ ID NO: 1 to 31. This level of amino acididentity may be seen at any section of the peptide, although it ispreferably the core region. The level of amino acid identity is over atleast 9 contiguous amino acids but it may be at least 10, 11, 12, 13,14, 15 or at least 16 or 17 amino acids, depending on the size of thepeptides of comparison. Accordingly, any of the above-specified levelsof identity may be across the entire length of sequence.

In connection with amino acid sequences, “sequence identity” refers tosequences which have the stated value when assessed using ClustalW(Thompson et al, Nucleic Acids Res. 1994 Nov 11; 22(22):4673-80) withthe following parameters: Pairwise alignment parameters—Method:accurate, Matrix: PAM, Gap open penalty: 10.00, Gap extension penalty:0.10; Multiple alignment parameters—Matrix: PAM, Gap open penalty:10.00, % identity for delay: 30, Penalize end gaps: on, Gap separationdistance: 0, Negative matrix: no, Gap extension penalty: 0.20,Residue—specific gap penalties: on, Hydrophilic gap penalties: on,Hydrophilic residues: GPSNDQEKR. Sequence identity at a particularresidue is intended to include identical residues which have simply beenderivatised.

A variant peptide may comprise 1, 2, 3, 4, 5 or more, or up to 10 aminoacid substitutions from any of SEQ ID NOS: 1 to 31. Substitutionvariants preferably involve the replacement of one or more amino acidswith the same number of amino acids and making conservative amino acidsubstitutions. For example, an amino acid may be substituted with analternative amino acid having similar properties, for example, anotherbasic amino acid, another acidic amino acid, another neutral amino acid,another charged amino acid, another hydrophilic amino acid, anotherhydrophobic amino acid, another polar amino acid, another aromatic aminoacid or another aliphatic amino acid. Some properties of the 20 mainamino acids which can be used to select suitable substituents are asfollows:

Ala aliphatic, hydrophobic, Met hydrophobic, neutral neutral Cys polar,hydrophobic, neutral Asn polar, hydrophilic, neutral Asp polar,hydrophilic, Pro hydrophobic, neutral charged (−) Glu polar,hydrophilic, Gln polar, hydrophilic, neutral charged (−) Phe aromatic,hydrophobic, Arg polar, hydrophilic, neutral charged (+) Gly aliphatic,neutral Ser polar, hydrophilic, neutral His aromatic, polar,hydrophilic, Thr polar, hydrophilic, neutral charged (+) Ile aliphatic,hydrophobic, Val aliphatic, hydrophobic, neutral neutral Lys polar,hydrophilic, Trp aromatic, hydrophobic, charged(+) neutral Leualiphatic, hydrophobic, Tyr aromatic, polar, neutral hydrophobic

Further variants include those in which instead of the naturallyoccurring amino acid the amino acid which appears in the sequence is astructural analog thereof. Amino acids used in the sequences may also bemodified, e.g. labelled, providing the function of the peptide is notsignificantly adversely affected.

Where the peptide has a sequence that varies from the sequence of any ofSEQ ID NOS: 1 to 31 or a fragment thereof, the substitutions may occuracross the full length of the sequence, within the sequence of any ofSEQ ID NOS: 1 to 31 or outside the sequence of any of SEQ ID NOS: 1 to31. For example, the variations described herein, such as additions,deletions, substitutions and modifications, may occur within thesequence of any of SEQ ID NOS: 1 to 31. A variant peptide may compriseor consist essentially of the amino acid sequence of any of SEQ ID NOS:1 to 31 in which one, two, three, four or more amino acid substitutionshave been made. A variant peptide may comprise a fragment of the parentprotein that is larger than any of SEQ ID NOS: 1 to 31. In thisembodiment, the variations described herein, such as substitutions andmodifications, may occur within and/or outside the sequence of any ofSEQ ID NOS: 1 to 31.

The variant peptides of the invention are 9 to 30 amino acids in lengthinclusive. Preferably, they may be from 9 to 20 or more preferably 13 to17 amino acids in length. The peptides may be the same length as thepeptide sequences in any one of SEQ ID NOS: 1 to 31.

The peptides may be chemically derived from the polypeptide allergen,for example by proteolytic cleavage or can be derived in an intellectualsense from the polypeptide allergen, for example by making use of theamino acid sequence of the polypeptide allergen and synthesisingpeptides based on the sequence. Peptides may be synthesised usingmethods well known in the art.

Where polypeptides comprise residues which are typically difficult topreserve during manufacture, these residues may be replaced. Forexample, glutamate spontaneously forms pyroglutamate in solutionparticularly when present at the N terminus of a peptide. Thus, residuesof the peptides of the invention which correspond to a glutamate orglutamine residue in the sequence of a native allergen protein sequencemay be replaced with pyroglutamate in the peptides of the invention whensuch a residue is present at the N terminus of a peptide.

The term “peptide” includes not only molecules in which amino acidresidues are joined by peptide (—CO—NH—) linkages but also molecules inwhich the peptide bond is reversed. Such retro-inverso peptidomimeticsmay be made using methods known in the art, for example such as thosedescribed in Meziere et al (1997) J. Immuno1.159, 3230-3237. Thisapproach involves making pseudopeptides containing changes involving thebackbone, and not the orientation of side chains. Meziere et al (1997)show that, at least for MHC class II and T helper cell responses, thesepseudopeptides are useful. Retro-inverse peptides, which contain NH—CObonds instead of CO—NH peptide bonds, are much more resistant toproteolysis. Similarly, the peptide bond may be dispensed withaltogether provided that an appropriate linker moiety which retains thespacing between the carbon atoms of the amino acid residues is used; itis particularly preferred if the linker moiety has substantially thesame charge distribution and substantially the same planarity as apeptide bond. It will also be appreciated that the peptide mayconveniently be blocked at its N- or C-terminus so as to help reducesusceptibility to exoproteolytic digestion. For example, the N-terminalamino group of the peptides may be protected by reacting with acarboxylic acid and the C-terminal carboxyl group of the peptide may beprotected by reacting with an amine. Other examples of modificationsinclude glycosylation and phosphorylation. Another potentialmodification is that hydrogens on the side chain amines of R or K may bereplaced with methylene groups (—NH₂→-NH(Me) or —N(Me)₂).

Analogues of peptides according to the invention may also includepeptide variants that increase or decrease the peptide's half-life invivo. Examples of analogues capable of increasing the half-life ofpeptides used according to the invention include peptoid analogues ofthe peptides, D-amino acid derivatives of the peptides, andpeptide-peptoid hybrids. A further embodiment of the variantpolypeptides used according to the invention comprises D-amino acidforms of the polypeptide. The preparation of polypeptides using D-aminoacids rather than L-amino acids greatly decreases any unwanted breakdownof such an agent by normal metabolic processes, decreasing the amountsof agent which needs to be administered, along with the frequency of itsadministration.

The peptides provided by the present invention may be derived fromsplice variants of the parent proteins encoded by mRNA generated byalternative splicing of the primary transcripts encoding the parentprotein chains. The peptides may also be derived from amino acidmutants, glycosylation variants and other covalent derivatives of theparent proteins which retain at least an MHC-binding property of theallergens. Exemplary derivatives include molecules wherein the peptidesof the invention are covalently modified by substitution, chemical,enzymatic, or other appropriate means with a moiety other than anaturally occurring amino acid. Further included are naturally occurringvariants of the parent proteins found in different mites. Such a variantmay be encoded by an allelic variant or represent an alternativesplicing variant.

Variants as described above may be prepared during synthesis of thepeptide or by post-production modification, or when the peptide is inrecombinant form using the known techniques of site-directedmutagenesis, random mutagenesis, or enzymatic cleavage and/or ligationof nucleic acids.

In accordance with the invention, the further peptides that thecomposition may comprise are preferably functional variants of any ofSEQ ID NOS: 1 to 31. That is, the peptides are preferably capable ofinducing an immune response. In particular, the peptides are preferablycapable of inducing cytokine production in ragweed allergic individuals.Typically, the composition of the invention will therefore comprise atleast one polypeptide or variant thereof which produces a cytokineresponse in greater than 45, 50, 55%, preferably 60% or 65% ofindividuals in a population of ragweed allergic individuals. The numberof individuals in a panel of ragweed allergic individuals may be anynumber greater than one, for example at least 20, 30, 40, 50, 80, or atleast 100 individuals. Preferably the composition comprises at leasttwo, three or most preferably four such peptides. Preferably thecytokine response is production of IL-13 or IFN-gamma. Cytokineproduction may be measured by any suitable method. Production of acytokine is typically considered to have occurred in response to apeptide if the level of cytokine produced in the presence of the peptideis at least 2, 3, 4 or 5 fold above the background level of saidcytokine that is produced in the absence of a stimulus (i.e. the levelproduced by the same individual in the absence of the peptide or anyother stimulus). Alternatively, production of a cytokine may beconsidered to have occurred if the amount of cytokine produced exceeds arecognised limit, typically 90, 95, or preferably 100 pg/ml, typicallyfrom a sample of approximately 1.25×10⁶ cells in 250 μl. Suitablemethods for measuring cytokine production typically include measuringthe cytokine release from peripheral blood mononuclear cells (PBMCs)from a taken sample from a subject. The sample is typically blood orserum. Cytokine release from PBMCs is measured after incubating thecells in the presence of a given peptide. Supernatants from theincubation mixture are then tested for the presence of a cytokine, usingany suitable assay, for example an ELISA, ELISPOT assay or flowcytometric assay. Particularly preferred methods include Multiplex beadarray assays as described in, for example de Jager et al; Clinical andDiagnostic Laboratory Immunology, 2003, Vol 10(1) p. 133-139.Typically,the composition may comprise at least one additional peptide or variantthereof that is not amongst the polypeptides already selected, upto atotal of thirteen different peptides, which produces a cytokine responsein greater than 30%, 35%, 40%, preferably 45% or 50% of individuals in apopulation of ragweed allergic individuals.

The composition may further comprise one or more additional peptides orvariants thereof that are not amongst the polypeptides already selected,upto a total of thirteen different peptides, which produce a cytokineresponse in greater than 10%, 15%, 20%, 25%, preferably 30% or 35% ofindividuals in a population of ragweed allergic individuals.

The composition may further comprise one or more additional peptideswhich induce release of IL-10. IL-10 is known as an immune modulatorwhich can shift T cell responses away from an allergic-type response. Asignificant IL-10 release may lead to induction of regulatory T cellswhich give rise to or improve toleration of the presence of the otherpeptides of the composition.

Preferably, said peptide may induce release of IL-10 in at least 35, 40,45, 50 or 55% of a population. In this embodiment, the peptide istherefore able to bind to a subset of MHC alleles which isrepresentative of an equivalent proportion of the sample population.Preferably, the peptides induce IL-10 release in 55% or more, 65% ormore, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or moreof a population.

“Induction of IL-10 release” is herein defined as a release which ismeasurable by methods commonly used in the art. Typically, the responseis measured in vitro using T cells obtained from the allergicindividuals. An “induction” is scored as an IL-10 level which is greaterthan that observed in a control sample where T cells are not exposed tothe peptide. In some embodiments, an induction of IL-10 release suitablefor tolerisation may be defined as an IL-10 release at least 35, 40, 45,50 or 55% of the average amount of IL-10 released in response to thewhole protein allergen of which the first polypeptide is a fragment. Itshould be understood that the protein allergen used as a comparison maybe the whole intact polypeptide or may be a truncated form thatcomprises the T cell epitopes which mediate immune response to theprotein allergen. Commonly, individual peptides derived from the proteinallergen will show an average IL-10 release that is much lower than thatobtained in response to the whole or truncated protein allergen asdefined above. However, individual peptides that show IL-10 release atleast 35, 40, 45, 50 or 55% of the IL-10 release to the whole ortruncated protein allergen may be particularly suitable tolerisingagents. The peptides may also display an average response that is thesame as, or greater than, the response observed in response to whole ortruncated protein allergen.

Alternatively the average level of IL-10 released may be measured inabsolute terms, in which case an average level above approximately 400,450, 500 or 550 pg/ml will be considered to be an induction, typicallyfrom a sample of approximately 1.25×10⁶ cells in 250 μl.

It should be understood that average may be the mean, median or mode ofthe individual IL-10 releases observed in the population. It should beunderstood that where an individual in the population displays anunusually low or unusually high IL-10 release in comparison to the othermembers of the population, they may be excluded from the average. Thismay allow for measurement of an average that is more representative ofthe responses shown in the population. The term ‘unusually low’ orunusually high' may refer to differentials of 10-fold or 20-fold ascompared to a more representative average of the IL-10 releases thatexcludes the individuals showing unusual IL-10 release characteristics.

Suitable variants capable of binding to TCRs may be derived empiricallyor selected according to known criteria. Within a single peptide thereare certain residues which contribute to binding within the MHC antigenbinding groove and other residues which interact with hypervariableregions of the T cell receptor (Allen et al (1987) Nature 327: 713-5).

Within the residues contributing to T cell receptor interaction, ahierarchy has been demonstrated which pertains to dependency of T cellactivation upon substitution of a given peptide residue. Using peptideswhich have had one or more T cell receptor contact residues substitutedwith a different amino acid, several groups have demonstrated profoundeffects upon the process of T cell activation. Evavold & Allen (1991)Nature 252: 1308-10) demonstrated the dissociation of T cellproliferation and cytokine production. In this in vitro model, a T cellclone specific for residues 64-76 of haemoglobin (in the context ofI-E^(k)), was challenged with a peptide analogue in which a conservativesubstitution of aspartic acid for glutamic acid had been made. Thissubstitution did not significantly interfere with the capacity of theanalogue to bind to I-E^(k).

Following in vitro challenge of a T cell clone with this analogue, noproliferation was detected although IL-4 secretion was maintained, aswas the capacity of the clone to help B cell responses. In a subsequentstudy the same group demonstrated the separation of T cell-mediatedcytolysis from cytokine production. In this instance, the formerremained unaltered while the latter was impaired. The efficacy ofaltered peptide ligands in vivo was initially demonstrated in a murinemodel of EAE (experimental allergic encephalomyelitis) by McDevitt andcolleagues (Smilek et al (1991) Proc Natl Acad Sci USA 88 : 9633-9637).In this model EAE is induced by immunisation with the encephalitogenicpeptide Ac1-11 of MBP (myelin basic protein). Substitution at positionfour (lysine) with an alanine residue generated a peptide which boundwell to its restricting element (Aα^(u)Aβ^(u)), but which wasnon-immunogenic in the susceptible PL/JxSJLF1 strain and which,furthermore prevented the onset of EAE when administered either beforeor after immunisation with the encephalitogenic peptide. Thus, residuescan be identified in peptides which affect the ability of the peptidesto induce various functions of T-cells.

Advantageously, peptides may be designed to favour T-cell proliferationand induction of desensitisation. Metzler and Wraith have demonstratedimproved tolerogenic capacity of peptides in which substitutionsincreasing peptide-MHC affinity have been made (Metzler & Wraith(1993)Int Immunol˜: 1159-65). That an altered peptide ligand can causelong-term and profound anergy in cloned T cells was demonstrated bySloan-Lancaster et al (1993) Nature 363: 156-9.

The compositions of the invention are capable of inducing a late phaseresponse in an individual that is sensitised to the allergens. The term“late phase response” includes the meaning as set forth in Allergy andAllergic Diseases (1997) A. B. Kay (Ed.), Blackwell Science, pp1113-1130. The late phase response may be any late phase response (LPR).Preferably, the peptides are capable of inducing a late asthmaticresponse (LAR) or a late rhinitic response, or a late phase skinresponse or a late phase ocular response. Whether or not a particularpeptide can give rise to a LPR can be determined using methods wellknown in the art; a particularly preferred method is that described inCromwell O, Durham S R, Shaw R J, Mackay J and Kay A B. Provocationtests and measurements of mediators from mast cells and basophils inasthma and allergic rhinitis. In: Handbook of Experimental Immunology(4) Chapter 127, Editor: Weir D M, Blackwell Scientific Publications,1986.

Thus, preferably, the individual peptides of the invention are able toinduce a LPR in an individual who has been sensitised to the allergens.Whether or not an individual has been sensitised to the allergens may bedetermined by well known procedures such as skin prick testing withsolutions of allergen extracts, induction of cutaneous LPRs, clinicalhistory, allergen challenge and radioallergosorbent test (RAST) formeasurement of allergen specific IgE. Whether or not a particularindividual is expected to benefit from treatment may be determined bythe physician based, for example, on such tests.

Desensitising or tolerising an individual to the allergens meansinhibition or dampening of allergic tissue reactions induced by theallergens in appropriately sensitised individuals. It has been shownthat T cells can be selectively activated, and then renderedunresponsive. Moreover the anergising or elimination of these T-cellsleads to desensitisation of the patient for a particular allergen. Thedesensitisation manifests itself as a reduction in response to anallergen or allergen-derived peptide, or preferably an elimination ofsuch a response, on second and further administrations of the allergenor allergen-derived peptide. The second administration may be made aftera suitable period of time has elapsed to allow desensitisation to occur;this is preferably any period between one day and several weeks. Aninterval of around two weeks is preferred.

Although the compositions of the invention are able to induce a LPR in aragweed allergic individual, it should be appreciated that when acomposition is used to treat a patient it is preferable that asufficiently low concentration of the composition is used such that noobservable LPR will occur but the response will be sufficient topartially desensitise the T cells such that the next (preferably higher)dose may be given, and so on. In this way the dose is built up to givefull desensitisation but often without ever inducing a LPR in thepatient. Although, the composition or peptide is able to do so at ahigher concentration than is administered.

The compositions of the invention preferably are capable of inducing alate phase response in 50% or more of a panel of ragweed allergicindividuals from the population. More preferably, the compositions arecapable of inducing a LPR in 55% or more, 60% or more, 65% or more, 70%or more, 75% or more, 80% or more, 85% or more, or 90% or more ofsensitized individuals in a panel. Whether or not the compositions areable to induce a LPR in a certain percentage of a panel of subjects canbe determined by methods which are well known in the art.

It will be understood that the peptides of the invention comprise a Tcell epitope that consists of a core 9 amino acids which are the minimalessential sequence required for MHC class II binding. However, thepeptides may also comprise additional residues flanking the core 9 aminoacids. The peptides may therefore comprise a region containing a T cellepitope, in which some residues may be modified without affecting thefunction of the epitope. Accordingly, functional variants of thepeptides as defined above include peptides which are altered to improvetheir solubility relative to the native sequence of the peptides.Improved solubility is advantageous for the tolerisation of subjects toallergens from which the peptides of the invention derive, sinceadministration of poorly soluble agents to subjects causes undesirable,non-tolerising inflammatory responses. The solubility of the peptidesmay be improved by altering the residues which flank the regioncontaining a T cell epitope. A peptide of the invention may beengineered to be more soluble such that it comprises:

-   i) N terminal to the residues of the peptide which flank a T cell    epitope: one to six contiguous amino acids corresponding to the two    to six contiguous amino acids immediately N terminal to said    residues in the sequence of the protein from which the peptide    derives; and/or-   ii) C terminal to the residues of the peptide which flank a T cell    epitope: one to six contiguous amino acids corresponding to the one    to six contiguous amino acids immediately C terminal to the said    residues in the sequence of the protein from which the peptide    derives; or-   iii) N and/or C terminal to the residues of the peptide which flank    a T cell epitope, at least one amino acid selected from arginine,    lysine, histidine, glutamate and aspartate.

Optionally, the peptides may additionally be engineered to be moresoluble such that:

-   i) any cysteine residues in the native sequence of the peptide are    replaced with serine or 2-aminobutyric acid; and/or-   ii) any residues at the N and/or C terminus of the native sequence    of the peptide, which are not comprised in a T cell epitope, are    deleted; and/or-   iii) any two consecutive amino acids comprising the sequence Asp-Gly    in the upto four amino acids at the N and/oror C terminus of the    native sequence of the peptide, which are not comprised in a T cell    epitope, are deleted.

Nucleic Acids and Vectors

The individual peptides that make up the compositions and products ofthe invention may be administered directly, or may be administeredindirectly by expression from an encoding sequence. For example, apolynucleotide may be provided that encodes a peptide of the invention,such as any of the peptides described above. A peptide of the inventionmay thus be produced from or delivered in the form of a polynucleotidewhich encodes, and is capable of expressing, it. Any reference herein tothe use, delivery or administration of a peptide of the invention isintended to include the indirect use, delivery or administration of sucha peptide via expression from a polynucleotide that encodes it.

The terms “nucleic acid molecule” and “polynucleotide” are usedinterchangeably herein and refer to a polymeric form of nucleotides ofany length, either deoxyribonucleotides or ribonucleotides, or analogsthereof. Non-limiting examples of polynucleotides include a gene, a genefragment, messenger RNA (mRNA), cDNA, recombinant polynucleotides,plasmids, vectors, isolated DNA of any sequence, isolated RNA of anysequence, nucleic acid probes, and primers. A polynucleotide of theinvention may be provided in isolated or purified form. A nucleic acidsequence which “encodes” a selected polypeptide is a nucleic acidmolecule which is transcribed (in the case of DNA) and translated (inthe case of mRNA) into a polypeptide in vivo when placed under thecontrol of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxy) terminus. Forthe purposes of the invention, such nucleic acid sequences can include,but are not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA,genomic sequences from viral or prokaryotic DNA or RNA, and evensynthetic DNA sequences. A transcription termination sequence may belocated 3′ to the coding sequence.

Polynucleotides of the invention can be synthesised according to methodswell known in the art, as described by way of example in Sambrook et al(1931, Molecular Cloning—a laboratory manual; Cold Spring Harbor Press).

The polynucleotide molecules of the present invention may be provided inthe form of an expression cassette which includes control sequencesoperably linked to the inserted sequence, thus allowing for expressionof the peptide of the invention in vivo in a targeted subject. Theseexpression cassettes, in turn, are typically provided within vectors(e.g., plasmids or recombinant viral vectors) which are suitable for useas reagents for nucleic acid immunization. Such an expression cassettemay be administered directly to a host subject. Alternatively, a vectorcomprising a polynucleotide of the invention may be administered to ahost subject. Preferably the polynucleotide is prepared and/oradministered using a genetic vector. A suitable vector may be any vectorwhich is capable of carrying a sufficient amount of genetic information,and allowing expression of a peptide of the invention.

The present invention thus includes expression vectors that comprisesuch polynucleotide sequences. Thus, the present invention provides avector for use in preventing or treating allergy to ragweeds bytolerisation comprising four or more polynucleotide sequences whichencode different polypeptides of the invention and optionally one ormore further polynucleotide sequences which encode differentpolypeptides as defined herein. The vector may comprise 4, 5, 6 or 7polynucleotide sequences which encode different polypeptides of theinvention.

Furthermore, it will be appreciated that the compositions and productsof the invention may comprise a mixture of polypeptides andpolynucleotides.

Accordingly, the invention provides a composition or product as definedherein, wherein in place of any one of the polypeptide is apolynucleotide capable of expressing said polypeptide.

Expression vectors are routinely constructed in the art of molecularbiology and may for example involve the use of plasmid DNA andappropriate initiators, promoters, enhancers and other elements, such asfor example polyadenylation signals which may be necessary, and whichare positioned in the correct orientation, in order to allow forexpression of a peptide of the invention. Other suitable vectors wouldbe apparent to persons skilled in the art. By way of further example inthis regard we refer to Sambrook et al.

Thus, a polypeptide of the invention may be provided by delivering sucha vector to a cell and allowing transcription from the vector to occur.Preferably, a polynucleotide of the invention or for use in theinvention in a vector is operably linked to a control sequence which iscapable of providing for the expression of the coding sequence by thehost cell, i.e. the vector is an expression vector.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, a given regulatory sequence, such as a promoter,operably linked to a nucleic acid sequence is capable of effecting theexpression of that sequence when the proper enzymes are present. Thepromoter need not be contiguous with the sequence, so long as itfunctions to direct the expression thereof. Thus, for example,intervening untranslated yet transcribed sequences can be presentbetween the promoter sequence and the nucleic acid sequence and thepromoter sequence can still be considered “operably linked” to thecoding sequence.

A number of expression systems have been described in the art, each ofwhich typically consists of a vector containing a gene or nucleotidesequence of interest operably linked to expression control sequences.These control sequences include transcriptional promoter sequences andtranscriptional start and termination sequences. The vectors of theinvention may be for example, plasmid, virus or phage vectors providedwith an origin of replication, optionally a promoter for the expressionof the said polynucleotide and optionally a regulator of the promoter. A“plasmid” is a vector in the form of an extrachromosomal geneticelement. The vectors may contain one or more selectable marker genes,for example an ampicillin resistance gene in the case of a bacterialplasmid or a resistance gene for a fungal vector. Vectors may be used invitro, for example for the production of DNA or RNA or used to transfector transform a host cell, for example, a mammalian host cell. Thevectors may also be adapted to be used in vivo, for example to allow invivo expression of the polypeptide.

A “promoter” is a nucleotide sequence which initiates and regulatestranscription of a polypeptide-encoding polynucleotide. Promoters caninclude inducible promoters (where expression of a polynucleotidesequence operably linked to the promoter is induced by an analyte,cofactor, regulatory protein, etc.), repressible promoters (whereexpression of a polynucleotide sequence operably linked to the promoteris repressed by an analyte, cofactor, regulatory protein, etc.), andconstitutive promoters. It is intended that the term “promoter” or“control element” includes full-length promoter regions and functional(e.g., controls transcription or translation) segments of these regions.

A polynucleotide, expression cassette or vector according to the presentinvention may additionally comprise a signal peptide sequence. Thesignal peptide sequence is generally inserted in operable linkage withthe promoter such that the signal peptide is expressed and facilitatessecretion of a polypeptide encoded by coding sequence also in operablelinkage with the promoter.

Typically a signal peptide sequence encodes a peptide of 10 to 30 aminoacids for example 15 to 20 amino acids. Often the amino acids arepredominantly hydrophobic. In a typical situation, a signal peptidetargets a growing polypeptide chain bearing the signal peptide to theendoplasmic reticulum of the expressing cell. The signal peptide iscleaved off in the endoplasmic reticulum, allowing for secretion of thepolypeptide via the Golgi apparatus. Thus, a peptide of the inventionmay be provided to an individual by expression from cells within theindividual, and secretion from those cells.

Alternatively, polynucleotides of the invention may be expressed in asuitable manner to allow presentation of a peptide of the invention byan MHC class II molecule at the surface of an antigen presenting cell.For example, a polynucleotide, expression cassette or vector of theinvention may be targeted to antigen presenting cells, or the expressionof encoded peptide may be preferentially stimulated or induced in suchcells.

Polynucleotides of interest may be used in vitro, ex vivo or in vivo inthe production of a peptide of the invention. Such polynucleotides maybe administered or used in the prevention or treatment of allergy bytolerisation.

Methods for gene delivery are known in the art. See, e.g., U.S. Pat.Nos. 5,399,346, 5,580,859 and 5,531,466. The nucleic acid molecule canbe introduced directly into the recipient subject, such as by standardintramuscular or intradermal injection; transdermal particle delivery;inhalation; topically, or by oral, intranasal or mucosal modes ofadministration. The molecule alternatively can be introduced ex vivointo cells that have been removed from a subject. For example, apolynucleotide, expression cassette or vector of the invention may beintroduced into APCs of an individual ex vivo. Cells containing thenucleic acid molecule of interest are re-introduced into the subjectsuch that an immune response can be mounted against the peptide encodedby the nucleic acid molecule. The nucleic acid molecules used in suchimmunization are generally referred to herein as “nucleic acidvaccines.”

The polypeptides, polynucleotides, vectors or cells of the invention maybe present in a substantially isolated form. They may be mixed withcarriers or diluents which will not interfere with their intended useand still be regarded as substantially isolated. They may also be in asubstantially purified form, in which case they will generally compriseat least 90%, e.g. at least 95%, 98% or 99%, of the proteins,polynucleotides, cells or dry mass of the preparation.

Antigen Presenting Cells (APCs)

The invention encompasses the use in vitro of a method of producing apopulation of APCs that present the peptides of the invention on theirsurface, that may be subsequently used in therapy. Such a method may becarried out ex vivo on a sample of cells that have been obtained from apatient. The APCs produced in this way therefore form a pharmaceuticalagent that can be used in the treatment or prevention of ragweed allergyby tolerisation. The cells should be accepted by the immune system ofthe individual because they derive from that individual. Delivery ofcells that have been produced in this way to the individual from whomthey were originally obtained, thus forms a therapeutic embodiment ofthe invention.

Formulations and Compositions

The peptides, polynucleotides, vectors and cells of the invention may beprovided to an individual either singly or in combination. Each moleculeor cell of the invention may be provided to an individual in anisolated, substantially isolated, purified or substantially purifiedform. For example, a peptide of the invention may be provided to anindividual substantially free from the other peptides.

Whilst it may be possible for the peptides, polynucleotides orcompositions according to the invention to be presented in raw form, itis preferable to present them as a pharmaceutical formulation. Thus,according to a further aspect of the invention, the present inventionprovides a pharmaceutical formulation for use in preventing or treatingallergy to ragweeds by tolerisation comprising a composition, vector orproduct according to the invention together with one or morepharmaceutically acceptable carriers or diluents and optionally one ormore other therapeutic ingredients. The carrier (s) must be ‘acceptable’in the sense of being compatible with the other ingredients of theformulation and not deleterious to the recipient thereof. Typically,carriers for injection, and the final formulation, are sterile andpyrogen free.

Formulation of a composition comprising the peptide, polynucleotides orcells of the invention can be carried out using standard pharmaceuticalformulation chemistries and methodologies all of which are readilyavailable to the reasonably skilled artisan.

For example, compositions containing one or more molecules or cells ofthe invention can be combined with one or more pharmaceuticallyacceptable excipients or vehicles. Auxiliary substances, such as wettingor emulsifying agents, pH buffering substances and the like, may bepresent in the excipient or vehicle. These excipients, vehicles andauxiliary substances are generally pharmaceutical agents that do notinduce an immune response in the individual receiving the composition,and which may be administered without undue toxicity. Pharmaceuticallyacceptable excipients include, but are not limited to, liquids such aswater, saline, polyethyleneglycol, hyaluronic acid and ethanol.Pharmaceutically acceptable salts can also be included therein, forexample, mineral acid salts such as hydrochlorides, hydrobromides,phosphates, sulfates, and the like; and the salts of organic acids suchas acetates, propionates, malonates, benzoates, and the like. A thoroughdiscussion of pharmaceutically acceptable excipients, vehicles andauxiliary substances is available in Remington's Pharmaceutical Sciences(Mack Pub. Co., N.J. 1991).

Such compositions may be prepared, packaged, or sold in a form suitablefor bolus administration or for continuous administration. Injectablecompositions may be prepared, packaged, or sold in unit dosage form,such as in ampoules or in multi-dose containers containing apreservative. Compositions include, but are not limited to, suspensions,solutions, emulsions in oily or aqueous vehicles, pastes, andimplantable sustained-release or biodegradable formulations. Suchcompositions may further comprise one or more additional ingredientsincluding, but not limited to, suspending, stabilizing, or dispersingagents. In one embodiment of a composition for parenteraladministration, the active ingredient is provided in dry (for e.g., apowder or granules) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration ofthe reconstituted composition. The pharmaceutical compositions may beprepared, packaged, or sold in the form of a sterile injectable aqueousor oily suspension or solution. This suspension or solution may beformulated according to the known art, and may comprise, in addition tothe active ingredient, additional ingredients such as the dispersingagents, wetting agents, or suspending agents described herein. Suchsterile injectable formulations may be prepared using a non-toxicparenterally-acceptable diluent or solvent, such as water or 1,3-butanediol, for example. Other acceptable diluents and solvents include, butare not limited to, Ringer's solution, isotonic sodium chloridesolution, and fixed oils such as synthetic mono-or di-glycerides. Otherparentally-administrable compositions which are useful include thosewhich comprise the active ingredient in microcrystalline form, in aliposomal preparation, or as a component of a biodegradable polymersystems. Compositions for sustained release or implantation may comprisepharmaceutically acceptable polymeric or hydrophobic materials such asan emulsion, an ion exchange resin, a sparingly soluble polymer, or asparingly soluble salt.

Alternatively, the peptides or polynucleotides of the present inventionmay be encapsulated, adsorbed to, or associated with, particulatecarriers. Suitable particulate carriers include those derived frompolymethyl methacrylate polymers, as well as PLG microparticles derivedfrom poly(lactides) and poly(lactide-co-glycolides). See, e.g., Jefferyet al. (1993) Pharm. Res. 10:362-368. Other particulate systems andpolymers can also be used, for example, polymers such as polylysine,polyarginine, polyornithine, spermine, spermidine, as well as conjugatesof these molecules.

The formulation of any of the peptides, polynucleotides or cellsmentioned herein will depend upon factors such as the nature of thesubstance and the method of delivery. Any such substance may beadministered in a variety of dosage forms. It may be administered orally(e.g. as tablets, troches, lozenges, aqueous or oily suspensions,dispersible powders or granules), parenterally, epicutaneously,subcutaneously, by inhalation, intravenously, intramuscularly,intrasternally, transdermally, intradermally, sublingually,instranasally, buccally or by infusion techniques. The substance mayalso be administered as suppositories. A physician will be able todetermine the required route of administration for each particularindividual.

The compositions of formulations of the invention will comprise asuitable concentration of each peptide/polynucleotide/cell to beeffective without causing adverse reaction. Typically, the concentrationof each peptide in the composition will be in the range of 0.03 to 200nmol/ml. More preferably in the range of 0.3 to 200 nmol/ml, 3 to 180nmol/ml, 10 to 150 nmol/ml or 30 to 120 nmol/ml. The composition orformulations should have a purity of greater than 95% or 98% or a purityof at least 99%.

In one embodiment, therefore, the peptides, polynucleotides, cells orcompositions of the invention are used for therapy in combination withone or more other therapeutic agents. The agents may be administeredseparately, simultaneously or sequentially. They may be administered inthe same or different compositions. Accordingly, in a method of theinvention, the subject may also be treated with a further therapeuticagent.

A composition may therefore be formulated which comprises a moleculeand/or cell of the invention and also one or more other therapeuticmolecules. A composition of the invention may alternatively be usedsimultaneously, sequentially or separately with one or more othertherapeutic compositions as part of a combined treatment.

Therapeutic Methods and Individual to be Treated

The present invention relates to peptides, polynucleotides, vectors andcells that are capable of desensitising or tolerising human individualsto the allergens described above and are therefore useful in theprevention or treatment of ragweed allergy. The invention providescompositions, products, vectors and formulations for use in preventingor treating allergy to ragweeds by tolerisation. The invention alsoprovides a method of tolerising or desensitizing a ragweed allergicindividual comprising administering, either singly or in combination thepolypeptides/polynucleotides/cells of the invention as described above.

The individual to be treated or provided with the composition orformulation of the invention is preferably human. It will be appreciatedthat the individual to be treated may be known to be sensitised to theallergens, at risk of being sensitised or suspected of being sensitised.The individual can be tested for sensitisation using techniques wellknown in the art and as described herein. Alternatively, the individualmay have a family history of allergy to ragweed. It may not be necessaryto test an individual for sensitisation to ragweed because theindividual may display symptoms of allergy when exposed to ragweed. Byexposure is meant proximity to, for example, a ragweed plant, or asubstance or product derived from a ragweed plant, or a substance orproduct containing or comprising either of the above. The substance orproduct derived from a ragweed plant is typically ragweed pollen. Byproximity is meant 10 metres or less, 5 metres or less, 2 metres orless, 1 metre or less, or 0 metres from the items described above.Symptoms of allergy can include itchy eyes, runny nose, breathingdifficulties, red itchy skin or rash.

The individual to be treated may be of any age. However, preferably, theindividual may be in the age group of 1 to 90, 5 to 60, 10 to 40, ormore preferably 18 to 35.

Preferably, the individual to be treated is from a population that hasMHC allele frequencies within the range of frequencies that arerepresentative of the Caucasian population. Reference population allelefrequencies for 11 common DRB1 allele families are shown in Table 1(Data from HLA Facts Book, Parham and Barber).

TABLE 1 DRB1 1 3 4 7 8 11 12 13 14 15 16 % 6.4 14.7 15.7 8.8 3.4 8.3 3.914.7 2.9 17.6 2.5 Reference 9.4 11.1 12.8 13.2 3.7 13.4 2.3 10.2 3.210.7 3.6 population %Reference frequencies were obtained by analysis of multiple studiesreporting frequencies and the figures shown are mean values. Preferablytherefore, the individual to be treated is from a population that hasequivalent MHC allele frequencies as the reference population for thealleles referred to Table 1 (such as for at least 1, 2, 3, 4, 5 or allof the alleles), for example within the ranges of those figures plus orminus 1, 2, 3, 5, 10, 15 or 20%.

Preferably the individual is from a population where the allelefrequencies of the following DRB1 alleles is:

-   4—at least 9%-   7—at least 10%-   11—at least 8%.

The individual may have had allergy to ragweeds for at least 2 weeks, 1month, 6 months, 1 year or 5 years. The individual may suffer from arash, nasal congestion, nasal discharge and/or coughing caused by theallergy. The individual may or may not have been administered with othercompositions/compounds which treat ragweed allergy.

The individual typically lives in a geographical region with a warm andmoist climate. The individual typically suffers from allergy to ragweedin a particular season. The season typically corresponds to theflowering season of ragweed, which is typically summer to Autumn,preferably late summer (for July to August in the Northern hemisphere)to early Autumn (September to October in the Northern hemisphere). Theragweed allergic individual is typically allergic to ragweed pollen.

Combination Immunotherapy

Since many individuals are allergic, or may require desensitizing toseveral polypeptide antigens, the current invention also provides meansof desensitizing individuals that are allergic to multiple antigens.“Tolerance” induced in an individual to a first polypeptide antigen orallergen can create in the individual a “tolergeneic environment”wherein inappropriate immune responses to other antigens can bedownregulated in order to provide tolerance to other antigens.

This finding means that individuals allergic to multiple allergens canbe treated in a greatly reduced time period, and that individualsseriously allergic to some allergens (e.g., peanuts) but more mildlyallergic to other allergens (e.g., cat dander) can benefit from atherapy wherein tolerance to the milder allergen is established and thenthis tolergeneic environment is used to provide tolerance to the other,more extreme allergen. In addition, individuals suffering from anautoimmune disorder who are additionally sensitised (or otherwiseimmune) to an unrelated antigen or allergen can benefit from a treatmentregime wherein tolerance to the unrelated antigen or allergen is firstestablished and then this tolergeneic environment is used to providetolerance to the autoantigen associated with the autoimmune disorder.

A method is therefore provided for desensitising a ragweed allergicindividual to ragweed allergen as described above and one or morefurther different polypeptide antigens. The method entails, in a firststep, administering to the individual a composition/product/formulation(primary composition) according to the invention as described herein andwherein the administration is carried out in a manner sufficient togenerate a hyporesponsive state against ragweed allergen. Once ahyporesponsive state has been established toward ragweed allergen, or atleast a shift toward desensitisation has occurred, the method entailsadministration of a secondary composition comprising a second, differentpolypeptide antigen to which the individual is to be sensitised.Administration of the secondary composition is carried out in such a wayas to take advantage of the tolergeneic environment established by useof the primary composition, where it is now possible to establishtolerance to the second, different polypeptide antigen. The secondarycomposition is coadministered with either the first primary compositionor a larger fragment of the allergen from which the primary compositionderives. By “coadministered” it is meant either the simultaneous orconcurrent administration, e.g., when the two are present in the samecomposition or administered in separate compositions at nearly the sametime but at different sites, as well as the delivery of polypeptideantigens in separate compositions at different times. For example, thesecondary composition may be delivered prior to or subsequent todelivery of the first composition at the same or a different site. Thetiming between deliveries can range from about several seconds apart toabout several minutes apart, several hours apart, or even several daysapart. Furthermore, different delivery methods can be employed.

The second polypeptide antigen is preferably an allergen different tothe ragweed allergen. Suitable allergens for use in the methods of theinvention can of course be obtained and/or produced using known methods.Classes of suitable allergens include, but are not limited to, otherragweed allergens, pollens, animal dander (especially cat dander),grasses, molds, dusts, antibiotics, stinging insect venoms, and avariety of environmental (including chemicals and metals), drug and foodallergens. Common tree allergens include pollens from cottonwood,popular, ash, birch, maple, oak, elm, hickory, and pecan trees; commonplant allergens include those from mugwort, ragweed, English plantain,sorrel-dock and pigweed; plant contact allergens include those frompoison oak, poison ivy and nettles; common grass allergens include ryegrass, Timothy, Johnson, Bermuda, fescue and bluegrass allergens; commonallergens can also be obtained from molds or fungi such as Alternaria,Fusarium, Hormodendrum, Aspergillus, Micropolyspora, Mucor andthermophilic actinomycetes; epidermal allergens can be obtained fromhouse or organic dusts (typically fungal in origin), or from animalsources such as feathers, and dog dander; common food allergens includemilk and cheese (diary), egg, wheat, nut (e.g., peanut), seafood (e.g.,shellfish), pea, bean and gluten allergens; common environmentalallergens include metals (nickel and gold), chemicals (formaldehyde,trinitrophenol and turpentine), Latex, rubber, fiber (cotton or wool),burlap, hair dye, cosmetic, detergent and perfume allergens; common drugallergens include local anesthetic and salicylate allergens; antibioticallergens include penicillin, tetracycline and sulfonamide allergens;and common insect allergens include bee, wasp and ant venom, andcockroach calyx allergens. Particularly well characterized allergensinclude, but are not limited to, the major cat allergen Fel dl, beevenom phospholipase A2 (PLA) (Akdis et al. (1996) J. Clin. Invest.98:1676-1683), birch pollen allergen Bet v 1 (Bauer et al. (1997) Clin.Exp. Immunol. 107:536-541), and the multi-epitopic recombinant grassallergen rKBG8.3 (Cao et al. (1997) Immunology 90:46-51). These andother suitable allergens are commercially available and/or can bereadily prepared as extracts following known techniques.

Preferably, the second polypeptide allergen is selected from the list ofallergen sequences and database accession numbers (NCBI Entrez accessionnumbers) below. NCBI is the National Center for Biotechnologyinformation and is a division of the US National Institutes of Health.The NCBI web site, from which access to the database may be sought, iswww.ncbi.nlm.nih.gov/. Allergen sequences and database accession numbers(NCBI Entrez accession numbers):

Dermatophagoides pteronyssinus

Der p 1 (SEQ ID NO: 47)MKIVLAIASLLALSAVYARPSSIKTFEEYKKAFNKSYATFEDEEAARKNFLESVKYVQSNGGAINHLSDLSLDEFKNRFLMSAEAFEHLKTQFDLNAETNACSINGNAPADDLRQMRTVTPIRMQGGCGSCWAFSGVAATESAYLAYRNQSLDLAEQELVDCASQHGCHGDTIPRGIEYIQHNGVVQESYYRYVAREQSCRRPNAQRFGISNYCQIYPPNVNKIREALAQTHSAIAVIIGIKDLDAFRHYDGRTIIQRDNGYQPNYHAVNIVGYSNAQGVDYWIVRNSWDTNWGDNGYGY FAANIDLMMIEEYPYVVILDer p 2 (SEQ ID NO: 48)MMYKILCLSLLVAAVARDQVDVKDCANHEIKKVLVPGCHGSEPCIIFIRGKPFQLEAVFEANQNTKTAKIEIKASIDGLEVDVPGIDPNACHYMKCPLVKGQQYDIKYTWNVPKIAPKSENVVVTVKVMGDDGVLACAIATHAKIRD Der p 3 (SEQ ID NO: 49)MIIYNILIVLLLAINTLANPILPASPNATIVGGEKALAGECPYQISLQSSSHFCGGTILDEYWILTAAHCVAGQTASKLSIRYNSLKHSLGGEKISVAKIFAHEKYDSYQIDNDIALIKLKSPMKLNQKNAKAVGLPAKGSDVKVGDQVRVSGWGYLEEGSYSLPSELRRVDIAVVSRKECNELYSKANAEVTDNMICGGDVANGGKDSCQGDSGGPVVDVKNNQVVGIVSWGYGCARKGYPGVYTRVGN FIDWIESKRSQ Der p 4(SEQ ID NO: 50) KYXNPHFIGXRSVITXLME Der p 5 (SEQ ID NO: 51)MKFIIAFFVATLAVMTVSGEDKKHDYQNEFDFLLMERIHEQIKKGELALFYLQEQINTHFEEKPTKEMKDKIVAEMDTIIAMIDGVRGVLDRLMQRKDLDIFEQYNLEMAKKSGDILERDLKKEEARVKKIEV Der p 6 (SEQ ID NO: 52)AIGXQPAAEAEAPFQISLMK Der p 7 (SEQ ID NO: 53)MMKLLLIAAAAFVAVSADPIHYDKITEEINKAVDEAVAAIEKSETFDPMKVPDHSDKFERHIGIIDLKGELDMRNIQVRGLKQMKRVGDANVKSEDGVVKAHLLVGVHDDVVSMEYDLAYKLGDLHPNTHVISDIQDFVVELSLEVSEEGNMTLTSFEVRQFANVVNHIGGLSILDPIFAVLSDVLTATQDTVRAEMTKV LAPAFKKELERNNQ Der p9(SEQ ID NO: 54) IVGGSNASPGDAVYQIAL

Dermatophagoides farinae

Der f 1 (SEQ ID NO: 55) MKFVLAIASLLVLTVYARPASIKTFEFKKAFNKNYATVEEEEVARKNFLESLKYVEANKGAINHLSDLSLDEFKNRYLMSAEAFEQLKTQFDLNAETSACRINSVNVPSELDLRSLRTVTPIRMQGGCGSCWAFSGVAATESAYLAYRNTSLDLSEQELVDCASQHGCHGDTIPRGIEYIQQNGVVEERSYPYVAREQRCRRPNSQHYGISNYCQIYPPDVKQIREALTQTHTAIAVIIGIKDLRAFQHYDGRTIIQHDNGYQPNYHAVNIVGYGSTQGDDYWIVRNSWDTTWGDSGYGYFQAGNNLMMIEQYPYVVIM Der f 2 (SEQ ID NO: 56)MISKILCLSLLVAAVVADQVDVKDCANNEIKKVMVDGCHGSDPCIIHRGKPFTLEALFDANQNTKTAKIEIKASLDGLEIDVPGIDTNACHFMKCPLVKGQQYDIKYTWNVPKIAPKSENVVVTVKLIGDNGVLACAI ATHGKIRD Der f 3(SEQ ID NO: 57) MMILTIVVLLAANILATPILPSSPNATIVGGVKAQAGDCPYQISLQSSSHFCGGSILDEYWILTAAHCVNGQSAKKLSIRYNTLKHASGGEKIQVAEIYQHENYDSMTIDNDVALIKLKTPMTLDQTNAKPVPLPAQGSDVKVGDKIRVSGWGYLQEGSYSLPSELQRVDIDVVSREQCDQLYSKAGADVSENMICGGDVANGGVDSCQGDSGGPVVDVATKQIVGIVSWGYGCARKGYPGVYTRVGNFVDWIESKRSQ Der f 4 (SEQ ID NO: 58)AVGGQDADLAEAPFQISLLK Der f 7 (SEQ ID NO: 59)MMKFLLIAAVAFVAVSADPIHYDKITEEINKAIDDAIAAIEQSETIDPMKVPDHADKFERHVGIVDFKGELAMRNIEARGLKQMKRQGDANVKGEEGIVKAHLLIGVHDDIVSMEYDLAYKLGDLHPTTHVISDIQDFVVALSLEISDEGNITMTSFEVRQFANVVNHIGGLSILDPIFGVLSDVLTAIFQDTVRKEMTKVLAPAFKRELEKN

Additional mite allergen sequences (NCBI entrez accession):

1170095; 1359436; 2440053; 666007; 487661; 1545803; 84702; 84699;625532; 404370; 1091577; 1460058; 7413; 9072; 387592.

Cat

Felis sequences (NCBI entrez accession):

539716; 539715; 423193; 423192; 423191; 423190; 1364213; 1364212;395407; 163827; 163823; 163825; 1169665; 232086; 1169666.

Latex

Hevea sequences:

Hev b 1 (SEQ ID NO: 60)MAEDEDNQQGQGEGLKYLGFVQDAATYAVTTFSNVYLFAKDKSGPLQPGVDIIEGPVKNVAVPLYNRFSYIPNGALKFVDSTVVASVTIIDRSLPPIVKDASIQVVSAIRAAPEAARSLASSLPGQTKILAKVFYGEN Hev b 3 (SEQ ID NO: 61)MAEEVEEERLKYLDFVRAAGVYAVDSFSTLYLYAKDISGPLKPGVDTIENVVKTVVTPVYYIPLEAVKFVDKTVDVSVTSLDGVVPPVIKQVSAQTYSVAQDAPRIVLDVASSVFNTGVQEGAKALYANLEPKAEQYAVITWRALNKLPLVPQVANVVVPTAVYFSEKYNDVVRGTTEQGYRVSSYLPLLPTEKITKVFG DEAS

Additional Hevea sequences (NCBI entrez accession):

3319923; 3319921; 3087805; 1493836; 1480457; 1223884; 3452147; 3451147;1916805; 232267; 123335; 2501578; 3319662; 3288200; 1942537; 2392631;2392630; 1421554; 1311006; 494093; 3183706; 3172534; 283243; 1170248;1708278; 1706547; 464775; 266312; 231586; 123337; 116359; 123062;2213877; 542013; 2144920; 1070656; 2129914; 2129913; 2129912; 100135;82026; 1076559; 82028; 82027; 282933; 280399; 100138; 1086972; 108697;1086976; 1086978; 1086978; 1086976; 1086974; 1086972; 913758; 913757;913756; 234388; 1092500; 228691; 1177405; 18839; 18837; 18835; 18833;18831; 1209317; 1184668; 168217; 168215; 168213; 168211; 168209; 348137.

Rye Grass

Lolium sequences:

126385 Lol p 1 (SEQ ID NO: 62)MASSSSVLLVVALFAVFLGS AHGIAKVPPGPNITAEYGDKWLDAKSTWYGKPTGAGPKDNGGACGYKNVDKAPFNGMTGCGNTPIFKDGRGCGSCFEIKCTKPESCSGEAVTVTITDDNEEPIAPYHFDLSGHAFGSMAKKGEEQNVRSAGELELQFRRVKCKYPDDTKPTFHVEKASNPNYLAIINKYVDGDGDVVAVDIKEKGKDKWIELKESWGAVWRIDTPDKLTGPFTVRYTTEGGTKSEFEDV IPEGWKADTSYSAK 126386 Lol p 2a (SEQ ID NO: 63)AAPVEFTVEKGSDEKNLALSIKYNKEGDSMAEVELKEHGSNEWLALKKNGDGVWEIKSDKPLKGPFNFRFVSEKGMRNVFDDVVPADFKVGTTYKPE 126387 Lol p 3(SEQ ID NO: 64) TKVDLTVEKGSDAKTLVLNIKYTRPGDTLAEVELRQHGSEEWEPMTKKGNLWEVKSAKPLTGPMNFRFLSKGGMKNVFDEVIPTAFTVGKTYTPEYN 2498581 Lol p 5a(SEQ ID NO: 65) MAVQKYTVALFLRRGPRGGPGRSYAADAGYTPAAAATPATPAATPAGGWREGDDRRAEAAGGRQRLASRQPWPPLPTPLRRTSSRSSRPPSPSPPRASSPTSAAKAPGLIPKLDTAYDVAYKAAEAHPRGQVRRLRHCPHRSLRVIAGALEVHAVKPATEEVLAAKIPTGELQIVDKIDAAFKIAATAANAAPTNDKFTVFESAFNKALNECTGGAMRPTSSSPPSRPRSSRPTPPPSPAAPEVKYAVFEAALTKAITAMTQAQKAGKPAAAAATAAATVATAAATAAAVLPPPLLVVQS LISLLIYY 2498582 Lol p 5b (SEQ ID NO: 66)MAVQKHTVALFLAVALVAGPAASYAADAGYAPATPATPAAPATAATPATPATPATPAAVPSGKATTEEQKLIEKINTAGFKAAVAAAAVVPPADKYKTFVETFGTATNKAFVEGLASGYADQSKNQLTSKLDAALKLAYEAAQGATPEAKYDAYVATLTEALRVIAGTLEVHAVKPAAEEVKVGAIPAAEVQLIDKVDAAYRTAATAANAAPANDKFTVFENTFNNAIKVSLGAAYDSYKFIPTLVAAVKQAYAAKQATAPEVKYTVSETALKKAVTAMSEAEKEATPAAAATATPTPAAATATATPAAAYATATPAAATATATPAAATATPAAAGGYKV 455288 Lol p isoform 9(SEQ ID NO: 67) MAVQKHTVALFLAVALVAGPAASYAADAGYAPATPATPAAPATAATPATPATPATPAAVPSGKATTEEQKLIEKINTAGFKAAVAAAAVVPPADKYKTFVETFGTATNKAFVEGLASGYADQSKNQLTSKLDAALKLAYEAAQGATPEAKYDAYVATLTEALRVIAGTLEVHAVKPAAEEVKVGAIPAAEVQLIDKVDAAYRTAATAANAAPANDKFTVFENTFNNAIKVSLGAAYDSYKFIPTLVAAVKQAYAAKQATAPEVKYTVSETALKKAVTAMSEAEKEATPAAAATATPTPAAATATATPAAAYATATPAAATATATPAAATATPAAAGGYKV 1582249 Lol p 11(SEQ ID NO: 68) DKGPGFVVTGRVYCDPCRAGFETNVSHNVEGATVAVDCRPFDGGESKLKAEATTDKDGWYKIEIDQDHQEEICEVVLAKSPDKSCSEIEEFRDRARVPLTSNXGIKQQGIRYANPIAFFRKEPLKECGGILQAY

Additional Lolium sequences (NCBI entrez accession):

135480; 417103; 687261; 687259; 1771355; 2388662; 631955; 542131;542130; 542129; 100636; 626029; 542132; 320616; 320615; 320614; 100638;100634; 82450; 626028; 100639; 283345; 542133; 1771353; 1763163; 310877;310875; 250525; 55317; 515377; 510911; 939932; 439950; 2718; 168316;168314; 485371; 2388664; 2832717; 2828273; 548867.

Olive Tree

Olive sequences

416610 Ole e 1 (SEQ ID NO: 69)EDIPQPPVSQFHIQGQVYCDTCRAGFITELSEFIPGASLRLQCKDKENGDVTFTEVGYTRAEGLYSMLVERDHKNEFCEITLISSGRKDCNEIPTEGWAKPSLKFKLNTVNGTTRTVNPLGFFKKEALPKCAQVYNKL GMYPPNM

Parietaria

Parietaria sequences:

2497750 Par j P2 (SEQ ID NO: 70)MRTVSMAALVVIAAALAWTSSAEPAPAPAPGEEACGKVVQDIMPCLHFVKGEEKEPSKECCSGTKKLSEEVKTTEQKREACKCIVRATKGISGIKNELVAEVPKKCDIKTTLPPITADFDCSKIQSTIFRGYY 1352506 Par j P5 (SEQ ID NO: 71)MVRALMPCLPFVQGKEKEPSKGCCSGAKRLDGETKTGPQRVHACECIQTAMKTYSDIDGKLVSEVPKHCGIVDSKLPPIDVNMDCKTVGVVPRQPQLPVSLRHGPVTGPSDPAHKARLERPQRVPPPAPEKA 1532056 Par j P8 (SEQ ID NO: 72)MRTVSMAALVVIAAALAWTSSAELASAPAPGEGPCGKVVHHIMPCLKFVKGEEKEPSKSCCSGTKKLSEEVKTTEQKREACKCIVAATKGISGIKNELVAEVPKKCGITTTLPPITADFDCSKIESTIFRGYY 1532058 Par j P9 (SEQ ID NO: 73)MRTVSAPSAVALVVIVAAGLAWTSLASVAPPAPAPGSEETCGTVVRALMPCLPFVQGKEKEPSKGCCSGAKRLDGETKTGLQRVHACECIQTAMKTYSDIDGKLVSEVPKHCGIVDSKLPPIDVNMDCKTLGVVPRQPQLPVSLRHGPVTGPSDPAHKARLERPQIRVPPPAPEKA 2497749 Par j P9 (SEQ ID NO: 74)MRTVSARSSVALVVIVAAVLVWTSSASVAPAPAPGSEETCGTVVGALMPCLPFVQGKEKEPSKGCCSGAKRLDGETKTGPQRVHACECIQTAMKTYSDIDGKLVSEVPKHCGIVDSKLPPIDVNMDCKTLGVLHYKGN 1086003 Par j 1 (SEQ ID NO: 75)MVRALMPCLPFVQGKEKEPSKGCCSGAKRLDGETKTGPQRVHACECIQTAMKTYSDIDGKLVSEVPKHCGIVDSKLPPIDVNMDCKTVGVVPRQPQLPVSLRHGPVTGPSRSRPPTKHGWRDPRLEFRPPHRKKPNPAFSTLG

Additional Parietaria sequences (NCBI entrez accession):

543659; 1836011; 1836010; 1311513; 1311512; 1311511; 1311510; 1311509;240971.

Timothy Grass

Phleum sequences:

Phl p 1 (SEQ ID NO: 76)MASSSSVLLVVVLFAVFLGSAYGIPKVPPGPNITATYGDKWLDAKSTWYGKPTGAGPKDNGGACGYKDVDKPPFSGMTGCGNTPIFKSGRGCGSCFEIKCTKPEACSGEPVVVHITDDNEEPIAPYHFDLSGHAFGAMAKKGDEQKLRSAGELELQFRRVKCKYPEGTKVTFHVEKGSNPNYLALLVKYVNGDGDVVAVDIKEKGKDKWIELKESWGAIWRIDTPDKLTGPFTVRYTTEGGTKTEAEDVI PEGWKADTSYESK Phl p 1(SEQ ID NO: 77) MASSSSVLLVVALFAVFLGSAHGIPKVPPGPNITATYGDKWLDAKSTWYGKPTAAGPKDNGGACGYKDVDKPPFSGMTGCGNTPIFKSGRGCGSCFEIKCTKPEACSGEPVVVHITDDNEEPIAAYHFDLSGIAFGSMAKKGDEQKLRSAGEVEIQFRRVKVKYPEGTKVTFHVEKGSNPNYLALLVKFSGDGDVVAVDIKEKGKDKWIALKESWGAIWRIDTPEVLKGPFTVRYTTEGGTKARAKDVIP EGWKADTAYESK Phl p 2(SEQ ID NO: 78) MSMASSSSSSLLAMAVLAALFAGAWCVPKVTFTVEKGSNEKHLAVLVKYEGDTMAEVELREHGSDEWVAMTKGEGGVWTFDSEEPLQGPFNFRFLTEKGMKNVFDDVVPEKYTIGATYAPEE Phl p 5 (SEQ ID NO: 79)ADLGYGGPATPAAPAEAAPAGKATTEEQKLIEKINDGFKAALAAAAGVPPADKYKTFVATFGAASNKAFAEGLSAEPKGAAESSSKAALTSKLDAAYKLAYKTAEGATPEAKYDAYVATLSEALRIIAGTLEVHAVKPAAEEVKVIPAGELQVIEKVDSAFKVAATAANAAPANDKFTVFEAAFNNAIKASTGGAYESYKFIPALEAAVKQAYAATVATAPEVKYTVFETALKKAFTAMSEAQKAAKPATEATATATAAVGAATGAATAATGGYKV Phl p 5 (SEQ ID NO: 80)ADLGYGGPATPAAPAEAAPAGKATTEEQKLIEKINDGFKAALAAAAGVPPADKYKTFVATFGAASNKAFAEGLSAEPKGAAESSSKAALTSKLDAAYKLAYKTAEGATPEAKYDAYVATLSEALRIIAGTLEVHAVKPAAEEVKVIPAGELQVIEKVDSAFKVAATAANAAPANDKFTVFEAAFNNAIKASTGGAYESYKFIPALEAAVKQAYAATVATAPEVKYTVFETALKKAITAMSEAQKAAKPATEATATATAAVGAATGAATAATGGYKV Phl p 5b (SEQ ID NO: 81)AAAAVPRRGPRGGPGRSYTADAGYAPATPAAAGAAAGKATTEEQKLIEDINTVGFKAAVAAAASVPAADKFKTFEAAFTSSSKAAAAKAPGLVPKLDAAYSVAYKAAVGATPEAKFDSFVASLTEALRVIAGALEVHAVKPVTEEPGMAKIPAGELQIIDKIDAAFKVAATAAATAPADDKFTVFEAAFNKAIKESTGGAYDTYKCIPSLEAAVKQAYAATVAAAPQVKYAVFEAALTKAITAMSEVQKVSQPATGAATVAAGAATTAAGAASGAATVAAGGYKV Phl p 5a (SEQ ID NO: 82)ADLGYGPATPAAPAAGYTPATPAAPAGADAAGKATTEEQKLIEKINTAGFKAALAGAGVQPADKYRTFVATFGPASNKAFAEGLSGEPKGAAESSSKAALTSKLDAAYKLAYKTAEGATPEAKYDAYVATLSEALRIIAGTLEVHAVKPAAEEVKVIPAGELQVIEKVDAAFKVAATAANAAPANDKFTVFEAAFNDEIKASTGGAYESYKFIPALEAAVKQAYAATVATAPEVKYTVFETALKKAITAMSEAQKAAKPAAAATATATAAVGAATGAATAATGGYKV Phl p 5 (SEQ ID NO: 83)MAVQKYTVALFLAVALVAGPAASYAADAGYAPATPAAAGAEAGKATTEEQKLIEDINTVGFKAAVAAAASVPAADKFKTFEAAFTSSSKAATAKAPGLVPKLDAAYSVSYKAAVGATPEAKFDSFVASLTEALRVIAGALEVHAVKPVTEEPGMAKIPAGELQIIDKIDAAFKVAATAAATAPADTVFEAAFNKAIKESTGGAYDTYKCIPSLEAAVKQAYAATVAAAPQVKYAVFEAALTKAITAMSEVQKVSQPATGAATVAAGAATTAAGAASGAATVAAGGYKV Phl p 5 (SEQ ID NO: 84)MAVQKYTVALFLAVALVAGPAASYAADAGYAPATPAAAGAEAGKATTEEQKLIEDINTVGFKAAVAAAASVPAADKFKTFEAAFTSSSKAATAKAPGLVPKLDAAYSVAYKAAVGATPEAKFDSFVASLTEALRVIAGALEVHAVKPVTEDPAWPKIPAGELQIIDKIDAAFKVAATAAATAPADDKFTVFEAAFNKAIKESTGGAYDTYKCIPSLEAAVKQAYAATVAAAPQVKYAVFEAALTKAITAMSEVQKVSQPATGAATVAAGAATTATGAASGAATVAAGGYKV Phl p 5 (SEQ ID NO: 85)ADAGYAPATPAAAGAEAGKATTEEQKLIEDINTVGFKAAVAAAASVPAADKFKTFEAAFTSSSKAATAKAPGLVPKLDAAYSVAYKAAVGATPEAKFDSFVASLTEALRVIAGALEVHAVKPVTEEPGMAKIPAGELQIIDKIDAAFKVAATAAATAPADDKFTVFEAAFNKAIKESTGGAYDTYKCIPSLEAAVKQAYAATVAAAPQVKYAVFEAALTKAITAMSEVQKVSQPATGAATVAAGAATTAA GAASGAATVAAGGYKVPhl p 5 (SEQ ID NO: 86)SVKRSNGSAEVHRGAVPRRGPRGGPGRSYAADAGYAPATPAAAGAEAGKATTEEQKLIEDINTVGFKAAVAAAASVPAADKFKTFEAAFTSSSKAATAKAPGLVPKLDAAYSVAYKAAVGATPEAKFDSFVASLTEALRVIAGALEVHAVKPVTEEPGMAKIPAGELQIIDKIDAAFKVAATAAATAPADDKFTVFEAAFNKAIKESTGGAYDTYKCIPSLEAAVKQAYAATVAAAPQVKYAVFEAALTKAITAMSEVQKVSQPATGAATVAAGAATTAAGAASGAATVAAGGYKV  Phl p 5 (SEQ ID NO: 87)MAVHQYTVALFLAVALVAGPAGSYAADLGYGPATPAAPAAGYTPATPAAPAGAEPAGKATTEEQKLIEKINAGFKAALAAAAGVPPADKYRTFVATFGAASNKAFAEGLSGEPKGAAESSSKAALTSKLDAAYKLAYKTAEGATPEAKYDAYVATVSEALRIIAGTLEVHAVKPAAEEVKVIPAGELQVIEKVDAAFKVAATAANAAPANDKFTVFEAAFNDAIKASTGGAYESYKFIPALEAAVKQAYAATVATAPEVKYTVFETALKKAITAMSEAQKAAKPAAAATATATAAVGAAT GAATAATGGYKV Phl p 5(SEQ ID NO: 88) ADLGYGGPATPAAPAEAAPAGKATTEEQKLIEKINDGFKAALAAAAGVPPADKYKTFVATFGAASNKAFAEGLSAEPKGAAESSSKAALTSKLDAAYKLAYKTAEGATPEAKYDAYVATLSEALRIIAGTLEVHAVKPAAEEVKVIPAGELQVIEKVDSAFKVAATAANAAPANDKFTVFEAAFNNAIKASTGGAYESYKFIPALEAAVKQAYAATVATAPEVKYTVFETALKKAFTAMSEAQKAAKPATEATATATAAVGAATGAATAATGGYKV Phl p5b (SEQ ID NO: 89)AAAAVPRRGPRGGPGRSYTADAGYAPATPAAAGAAAGKATTEEQKLIEDINTVGFKAAVAAAASVPAADKFKTFEAAFTSSSKAAAAKAPGLVPKLDAAYSVAYKAAVGATPEAKFDSFVASLTEALRVIAGALEVHAVKPVTEEPGMAKIPAGELQIIDKIDAAFKVAATAAATAPADDKFTVFEAAFNKAIKESTGGAYDTYKCIPSLEAAVKQAYAATVAAAPQVKYAVFEAALTKAITAMSEVQKVSQPATGAATVAAGAATTAAGAASGAATVAAGGYKV Phl p5a (SEQ ID NO: 90)ADLGYGPATPAAPAAGYTPATPAAPAGADAAGKATTEEQKLIEKMAGFKAALAGAGVQPADKYRTFVATFGPASNKAFAEGLSGEPKGAAESSSKAALTSKLDAAYKLAYKTAEGATPEAKYDAYVATLSEALRIIAGTLEVHAVKPAAEEVKVIPAGELQVIEKVDAAFKVAATAANAAPANDKFTVFEAAFNDEIKASTGGAYESYKFIPALEAAVKQAYAATVATAPEVKYTVFETALKKAITAMSEAQKAAKPAAAATATATAAVGAATGAATAATGGYKV Phl p 5 (SEQ ID NO: 91)AVPRRGPRGGPGRSYAADAGYAPATPAAAGAEAGKATTEEQKLIEDINVGFKAAVAAAASVPAGDKFKTFEAAFTSSSKAATAKAPGLVPKLDAAYSVAYKAAVGATPEAKFDSFVASLTEALRVIAGALEVHAVKPVTEEPGMAKIPAGELQIIDKIDAAFKVAATAAATAPADDKFTVFEAAFNKAIKESTGGAYDTYKCIPSLEAAVKQAYAATVAAAPQVKYAVFEAALTKAITAMSEVQKVSQPATGAATVAAGAATTATGAASGAATVAAGGYKV Phl p 5b (SEQ ID NO: 92)MAVPRRGPRGGPGRSYTADAGYAPATPAAAGAAAGKATTEEQKLIEDINVGFKAAVAARQRPAADKFKTFEAASPRHPRPLRQGAGLVPKLDAAYSVAYKAAVGATPEAKFDSFVASLTEALRVIAGALEVHAVKPVTEEPGMAKIPAGELQIIDKIDAAFKVAATAAATAPADDKFTVFEAAFNKAIKESTGGAYDTYKCIPSLEAAVKQAYAATVAAAAEVKYAVFEAALTKAITAMSEVQKVSQPATGAATVAAGAATTAAGAASGAATVAAGGYKV Phl p 5 (SEQ ID NO: 93)MAVHQYTVALFLAVALVAGPAASYAADLGYGPATPAAPAAGYTPATPAAPAEAAPAGKATTEEQKLIEKINAGFKAALAAAAGVQPADKYRTFVATFGAASNKAFAEGLSGEPKGAAESSSKAALTSKLDAAYKLAYKTAEGATPEAKYDAYVATLSEALRIIAGTLEVHAVKPAAEEVKVIPAGELQVIEKVDAAFKVAATAANAAPANDKFTVFEAAFNDAIKASTGGAYESYKFIPALEAAVKQAYAATVATAPEVKYTVFETALKKAITAMSEAQKAAKPAAAATATATAAVGAAT GAATAATGGYKV Phl p 5(SEQ ID NO: 94) EAPAGKATTEEQKLIEKINAGFKAALARRLQPADKYRTFVATFGPASNKAFAEGLSGEPKGAAESSSKAALTSKLDAAYKLAYKTAEGATPEAKYDAYVATLSEALRIIAGTLEVHAVKPAAEEVKVIPAAELQVIEKVDAAFKVAATAANAAPANDKFTVFEAAFNDEIKASTGGAYESYKFIPALEAAVKQAYAATVATAPEVKYTVFETALKKAITAMSEAQKAAKPPPLPPPPQPPPLAATGAATA ATGGYKV Phl p 5(SEQ ID NO: 95) MAVHQYTVALFLAVALVAGPAASYAADLGYGPATPAAPAAGYTPATPAAPAEAAPAGKATTEEQKLIEKINAGFKAALAAAAGVQPADKYRTFVATFGAASNKAFAEGLSGEPKGAAESSSKAALTSKLDAAYKLAYKTAEGATPEAKYDAYVATLSEALRIIAGTLEVHAVKPAAEEVKVIPAGELQVIEKVDAAFKVAATAANAAPANDKFTVFEAAFNDAIKASTGGAYESYKFIPALEAAVKQAYAATVATAPEVKYTVFETALKKAITAMSEAQKAAKPAAAATATATAAVGAAT GAATAATGGYKV Phl p 5b(SEQ ID NO: 96) MAVPRRGPRGGPGRSYTADAGYAPATPAAAGAAAGKATTEEQKLIEDINVGFKAAVAARQRPAADKFKTFEAASPRHPRPLRQGAGLVPKLDAAYSVAYKAAVGATPEAKFDSFVASLTEALRVIAGALEVHAVKPVTEEPGMAKIPAGELQIIDKIDAAFKVAATAAATAPADDKFTVFEAAFNKAIKESTGGAYDTYKCIPSLEAAVKQAYAATVAAAAEVKYAVFEAALTKAITAMSEVQKVSQPATGAATVAAGAATTAAGAASGAATVAAGGYKV Phl p 5a (SEQ ID NO: 97)ADLGYGPATPAAPAAGYTPATPAAPAGADAAGKATTEEQKLIEKINTAGFKAALAGAGVQPADKYRTFVATFGPASNKAFAEGLSGEPKGAAESSSKAALTSKLDAAYKLAYKTAEGATPEAKYDAYVATLSEALRIIAGTLEVHAVKPAAEEVKVIPAGELQVIEKVDAAFKVAATAANAAPANDKFTVFEAAFNDEIKASTGGAYESYKFIPALEAAVKQAYAATVATAPEVKYTVFETALKKAITAMSEAQKAAKPPPLPPPPQPPPLAATGAATAATGGYKV Phl p 5 (SEQ ID NO: 98)MAVHQYTVALFLAVALVAGPAASYAADLGYGPATPAAPAAGYTPATPAAPAEAAPAGKATTEEQKLIEKINAGFKAALAAAAGVQPADKYRTFVATFGAASNKAFAEGLSGEPKGAAESSSKAALTSKLDAAYKLAYKTAEGATPEAKYDAYVATLSEALRIIAGTLEVHAVKPAAEEVKVIPAGELQVIEKVDAAFKVAATAANAAPANDKFTVFEAAFNDAIKASTGGAYESYKFIPALEAAVKQAYAATVATAPEVKYTVFETALKKAITAMSEAQKAAKPAAAATATATAAVGAAT GAATAATGGYKV Phl p 6(SEQ ID NO: 99) MAAHKFMVAMFLAVAVVLGLATSPTAEGGKATTEEQKLIEDVNASFRAAMATTANVPPADKYKTFEAAFTVSSKRNLADAVSKAPQLVPKLDEVYNAAYNAADHAAPEDKYEAFVLHFSEALRIIAGTPEVHAVKPGA Phl p 6 (SEQ ID NO: 100)SKAPQLVPKLDEVYNAAYNAADHAAPEDKYEAFVLHFSEALHIIAGTPEV HAVKPGA  Phl p 6(SEQ ID NO: 101) ADKYKTFEAAFTVSSKRNLADAVSKAPQLVPKLDEVYNAAYNAADHAAPEDKYEAFVLHFSEALHIIAGTPEVHAVKPGA Phl p 6 (SEQ ID NO: 102)TEEQKLIEDVNASFRAAMATTANVPPADKYKTLEAAFTVSSKRNLADAVSKAPQLVPKLDEVYNAAYNAADHAAPEDKYEAFVLHFSEALRIIAGTPEVH AVKPGA Phl p 6(SEQ ID NO: 103) MAAHKFMVAMFLAVAVVLGLATSPTAEGGKATTEEQKLIEDINASFRAAMATTANVPPADKYKTFEAAFTVSSKRNLADAVSKAPQLVPKLDEVYNAAYNAADHAAPEDKYEAFVLHFSEALHIIAGTPEVHAVKPGA Phl p 6 (SEQ ID NO: 104)MVAMFLAVAVVLGLATSPTAEGGKATTEEQKLIEDVNASFRAAMATTANVPPADKYKTFEAAFTVSSKRNLADAVSKAPQLVPKLDEVYNAAYNAADHAAPEDKYEAFVLHFSEALRIIAGTPEVHAVKPGA Phl p 7 (SEQ ID NO: 105)MADDMERIFKRFDTNGDGKISLSELTDALRTLGSTSADEVQRMMAEIDTDGDGFIDFNEFISFCNANPGLMKDVAKVF  Phl p 11 (SEQ ID NO: 106)MSWQTYVDEHLMCEIEGHHLASAMLGHDGTVWAQSADFPQFKPEEITGIMKDFDEPGHLAPTGMFVAGAKYMVIQGEPGRVIRGKKGAGGITIKKTGQALVVGIYDEPMTPGQCNMVVERLGDYLVEQGM

Additional Phleum sequences (NCBI entrez accession):

458878; 548863; 2529314; 2529308; 2415702; 2415700; 2415698; 542168;542167; 626037; 542169; 541814; 542171; 253337; 253336; 453976; 439960 .

Wasp (and Related)

Vespula sequences:

465054 ALLERGEN VES V 5 (SEQ ID NO: 107)MEISGLVYLIIIVTIIDLPYGKANNYCKIKCLKGGVHTACKYGSLKPNCGNKVVVSYGLTKQEKQNLKEHNDFRQKIARGLETRGNPGPQPPAKNMKNLVWNDELAYVAQVWANQCQYGHDTCRDVAKYQVGQNVALTGSTAAKYDDPVKLVKMWEDEVKDYNPKKKFSGNDFLKTGHYTQMVWANTKEVGCGSIKYIQEKWHKHYLVCNYGPSGNFMNEELYQTK 1709545 ALLERGEN VES M 1 (SEQ ID NO: 108)GPKCPFNSDTVSIIIETRENRNRDLYTLQTLQNHPEFKKKTITRPVVFITHGFTSSASEKNFINLAKALVDKDNYMVISIDWQTAACTNEYPGLKYAYYPTAASNTRLVGQYIATITQKLVKDYKISMANIRLIGHSLGAHVSGFAGKRVQELKLGKYSEIIGLDPARPSFDSNHCSERLCETDAEYVQIIHTSNYLGTEKILGTVDFYMNNGKNNPGCGRFFSEVCSHTRAVIYMAECIKHECCLIGIPRSKSSQPISRCTKQECVCVGLNAKKYPSRGSFYVPVESTAPFCNNKGKII1352699 ALLERGEN VES V 1 (SEQ ID NO: 109)MEENMNLKYLLLFVYFVQVLNCCYGHGDPLSYELDRGPKCPFNSDTVSIIIETRENRNRDLYTLQTLQNHPEFKKKTITRPVVFITHGFTSSASETNFINTLAKALVDKDNYMVISIDWQTAACTNEAAGLKYLYYPTAARNTRLVGQYIATITQKLVKHYKISMANIRLIGHSLGAHASGFAGKKVQELKLGKYSEIIGLDPARPSFDSNHCSERLCETDAEYVQIIHTSNYLGTEKTLGTVDFYMNNGKNQPGCGRFFSEVCSHSRAVIYMAECIKHECCLIGIPKSKSSQPISSCTKQECVCVGLNAKKYPSRGSFYVPVESTAPFCNNKGKII  1346323 ALLERGEN VES V 2(SEQ ID NO: 110) SERPKRVFNIYWNVPTFMCHQYDLYFDEVTNFNIKRNSKDDFQGDKIAWYDPGEFPALLSLKDGKYKKRNGGVPQEGNITIHLQKFIENLDKIYPNRNFSGIGVIDFERWRPIFRQNWGNMKIHKNFSIDLVRNEHPTWNKKMIELEASKRFEKYARFFMEETLKLAKKTRKQADWGYYGYPYCFNMSPNNLVPECDVTAMHENDKMSWLFNNQNVLLPSVYVRQELTPDQRIGLVQGRVKEAVRISNNLKHSPKVLSYWWYVYQDETNTFLTETDVKKTFQEIVINGGDGIIIWGSSSDVNSLSKCKRLQDYLLTVLGPIAINIVTEAVN 549194 ALLERGEN VES VI (SEQ ID NO: 111)5KVNYCKIKCLKGGVHTACKYGTSTKPNCGKMVVKAYGLTEAEKQEILKVHNDFRQKVAKGLETRGNPGPQPPAKNMNNLVWNDELANIAQVWASQCNYGHDTCKDTEKYPVGQNIAKRSTTAALFDSPGKLVKMWENEVKDFNPNIEWSKNNLKKTGHYTQMVWAKTKEIGCGSVKYVKDEWYTHYLVCNYGPSGNFRN EKLYEKK 

Additional vespula sequences (NCBI entrez accession): 549193; 549192;549191; 549190; 549131; 117414; 126761; 69576; 625255; 627131; 627188;627187; 482382; 112561; 627186; 627185; 1923233; 317645; 317647; 745570;225764; 162551.

Tree allergen sequences (mainly birch) sequences:

114922 Bet v 1 (SEQ ID NO: 112)MGVFNYETETTSVIPAARLFKAFILDGDNLFPKVAPQAISSVENIEGNGGPGTIKKISFPEGFPFKYVKDRVDEVDHTNFKYNYSVIEGGPIGDTLEKISNEIKIVATPDGGSILKISNKYHTKGDHEVKAEQVKASKEMGETLLRAVES YLLAHSDAYN 130975 Bet v 2 (SEQ ID NO: 113)MS WQTYVDEHLMCDIDGQASNSLASAIVGHDGSVWAQSSSFPQFKPQEITGIMKDFEEPGHLAPTGLHLGGIKYMVIQGEAGAVIRGKKGSGGITIKKTGQALVFGIYEEPVTPGQCNMVVERLGDYLIDQGL 1168696 Bet v 3 (SEQ ID NO: 114)MPCSTEAMEKAGHGHASTPRKRSLSNSSFRLRSESLNTLRLRRIFDLFDKNSDGIITVDELSRALNLLGLETDLSELESTVKSFTREGNIGLQFEDFISLHQSLNDSYFAYGGEDEDDNEEDMRKSILSQEEADSFGGFKVFDEDGDGYISARELQMVLGKLGFSEGSEIDRVEKMIVSVDSNRDGRVDFFEFKDMMRSV LVRSS 809536 Bet v 4(SEQ ID NO: 115) MADDHPQDKAERERIFKRFDANGDGKISAAELGEALKTLGSITPDEVKHMMAEIDTDGDGFISFQEFTDFGRANRGLLKDVAKIF 543675 Que a I-Quercus alba =oak trees (fragment) (SEQ ID NO: 116) GVFTXESQETSVIAPAXLFKALFL543509 Car b I-Carpinus betulus = hornbeam trees  (fragment)(SEQ ID NO: 117) GVFNYEAETPSVIPAARLFKSYVLDGDKLIPKVAPQMXK543491 Aln g I-Alnus glutinosa = alder trees  (fragment)(SEQ ID NO: 118) GVFNYEAETPSVIPAARLFKAHLDGDKLLPKVAPEAVSSVENI1204056 Rubisco (SEQ ID NO: 119)VQCMQVWPPLGLKKFETLSYLPPLSSEQLAKEVDYLLRKNLIPCLEFELEHGFVYREHNRSPGYYDGRYWTMWKLPMFGCNDSSQVLKELEECKKAYPSA FIRIIGFDDK

Additional tree allergen sequences (NCBI entrez accession number):

131919; 128193; 585564; 1942360; 2554672; 2392209; 2414158; 1321728;1321726; 1321724; 1321722; 1321720; 1321718; 1321716; 1321714; 1321712;3015520; 2935416; 464576; 1705843; 1168701; 1168710; 1168709; 1168708;1168707; 1168706; 1168705; 1168704; 1168703; 1168702; 1842188; 2564228;2564226; 2564224; 2564222; 2564220; 2051993; 1813311; 1536831; 534910;534900; 534318; 1340000; 1339998; 2149808; 66207; 2129477; 1076249;1076247; 629480; 481805; 81443; 1361968; 1361967; 1361966; 1361965;1361964; 1361963; 1361962; 1361961; 1361960; 1361959; 320546; 629483;629482; 629481; 541804; 320545; 81444; 541814:; 629484; 474911; 452742;1834387; 298737; 298736; 1584322; 1584321; 584320; 1542873; 1542871;1542869; 1542867; 1542865; 1542863; 1542861; 1542859; 1542857; 1483232;1483230; 1483228; 558561; 551640; 488605; 452746; 452744; 452740;452738; 452736; 452734; 452732; 452730; 452728; 450885; 17938; 17927;17925; 17921; 297538; 510951; 231331; 231329; 166953 .

Peanut

Peanut sequences

1168391 Ara h 1 (SEQ ID NO: 120)MRGRVSPLMLLLGILVLASVSATHAKSSPYQKKTENPCAQRCLQSCQQEPDDLKQKACESRCTKLEYDPRCVYDPRGHTGTTNQRSPPGERTRGRQPGDYDDDRRQPRREEGGRWGPAGPREREREEDWRQPREDWRRPSHQQPRKIRPEGREGEQEWGTPGSHVREETSRNNPFYFPSRRFSTRYGNQNGRIRVLQRFDQRSRQFQNLQNHRIVQIEAKPNTLVLPKHADADNILVIQQGQATVTVANGNNRKSFNLDEGHALRIPSGFISYILNRHDNQNLRVAKISMPVNTPGQFEDFFPASSRDQSSYLQGFSRNTLEAAFNAEFNEIRRVLLEENAGGEQEERGQRRWSTRSSENNEGVIVKVSKEHVEELTKHAKSVSKKGSEEEGDITNPINLREGEPDLSNNFGKLFEVKPDKKNPQLQDLDMMLTCVEIKEGALMLPHFNSKAMVIVVVNKGTGNLELVAVRKEQQQRGRREEEEDEDEEEEGSNREVRRYTARLKEGDVFIMPAAHPVAINASSELHLLGFGINTAENNHRIFLAGDKDNVIDQIEKQAKDLAFPGSGEQVEKLIKNQKESHFVSARPQSQSQSPSSPEKESPEKEDQEEENQGGKGPLLSILKAFN

Ragweed

Ambrosia sequences

113478 Amb a 1 (SEQ ID NO: 121)MGIKHCCYILYFTLALVTLLQPVRSAEDLQQILPSANETRSLTTCGTYNIIDGCWRGKADWAENRKALADCAQGFAKGTIGGKDGDIYTVTSELDDDVANPKEGTLRFGAAQNRPLWIIFARDMVIRLDRELAINNDKTIDGRGAKVEIINAGFAIYNVKNIIIHNIIMHDIVVNPGGLIKSHDGPPVPRKGSDGDAIGISGGSQIWIDHCSLSKAVDGLIDAKHGSTHFTVSNCLFTQHQYLLLFWDFDERGMLCTVAFNKFTDNVDQRMPNLRHGFVQVVNNNYERWGSYALGGSAGPTILSQGNRFLASDIKKEVVGRYGESAMSESINTWNWRSYMDVFENGAIFVPSGVDPVLTPEQNAGMIPAEPGEAVLRLTSSAGVLSCQPGAPC 113479 Amb a2(SEQ ID NO: 122) MGIKHCCYILYFTLALVTLVQAGRLGEEVDILPSPNDTRRSLQGCEAHNIIDKCWRCKPDWAENRQALGNCAQGFGKATHGGKWGDIYMVTSDQDDDVVNPKEGTLRFGATQDRPLWIIFQRDMITYLQQEMVVTSDKTIDGRGAKVELVYGGITLMNVKNVIIHNIDIHDVRVLPGGRIKSNGGPAIPRHQSDGDAIHVTGSSDIWIDHCTLSKSFDGLVDVNWGSTGVTISNCKFTHHEKAVLLGASDTHFQDLKMHVTLAYNIFTNTVHERMPRCRFGFFQIVNNFYDRWDKYAIGGSSNPTILSQGNKFVAPDFIYKKNVCLRTGAQEPEWMTWNWRTQNDVLENGAIFVASGSDPVLTAEQNAGMMQAEPGDMVPQLTMNAGVLTCSPGAPC 113477 Amb a 1.3(SEQ ID NO: 123) MGIKQCCYILYFTLALVALLQPVRSAEGVGEILPSVNETRSLQACEALNIIDKCWRGKADWENNRQALADCAQGFAKGTYGGKWGDVYTVTSNLDDDVANPKEGTLRFAAAQNRPLWITKNDMVINLNQELVVNSDKTIDGRGVKVEIINGGLTLMNVKNIIIHNINTIHDVKVLPGGMIKSNDGPPILRQASDGDTINVAGSSQIWIDHCSLSKSFDGLVDVTLGSTHVTISNCKFTQQSKAILLGADDTHVQDKGMLATVAFNMFTDNVDQRMPRCRFGFFQVVNNNYDRWGTYAIGGSSAPTILCQGNRFLAPDDQIKKNVLARTGTGAAESMAWNWRSDKDLLENGATVTSGSDPVLTPVQSAGMIPAEPGEAAIKLTSSAGVFSCHPGAPC 113476 Amb a 1.2(SEQ ID NO: 124) MGIKHCCYILYFTLALVTLLQPVRSAEDVEEFLPSANETRRSLKACEAHNIIDKCWRCKADWANNRQALADCAQGFAKGTYGGKHGDVYTVTSDKDDDVANPKEGTLRFAAAQNRPLWITKRNMVIFILNQELVVNSDKTIDGRGVKVNIVNAGLTLMNVKNIIIHNINTIHDIKVCPGGMIKSNDGPPILRQQSDGDAINVAGSSQIWIDHCSLSKASDGLLDITLGSSHVTVSNCKFTQHQFVLLLGADDTHYQDKGMLATVAFNMFTDHVDQRMPRCRFGFFQVVNNNYDRWGTYAIGGSSAPTILSQGNRFFAPDDIIKKNVLARTGTGNAESMSWNWRTDRDLLENGAIFLPSGSDPVLTPEQKAGMIPAEPGEAVLRLTSSAGVLSCHQGAPC 113475 Amb a 1.1(SEQ ID NO: 125) MGIKHCCYILYFTLALVTLLQPVRSAEDLQEILPVNETRRLTTSGAYNIIDGCWRGKADWAENRKALADCAQGFGKGTVGGKDGDIYTVTSELDDDVANPKEGTLRFGAAQNRPLWITERDMVIRLDKEMVVNSDKTIDGRGAKVEIINAGFTLNGVKNVIIHNINMHDVKVNPGGLIKSNDGPAAPRAGSDGDAISISGSSQIWIDHCSLSKSVDGLVDAKLGTTRLTVSNSLFTQHQFVLLFGAGDENIEDRGMLATVAFNTFTDNVDQRMPRCRHGFFQVVNNNYDKWGSYAIGGSASPTILSQGNRFCAPDERSKKNVLGRHGEAAAESMKWNWRTNKDVLENGATVASGVDPVLTPEQSAGMIPAEPGESALSLTSSAGVLSCQPGAPC

Cedar sequences

493634 Cry j IB precursor (SEQ ID NO: 126)MDSPCLVALLVFSFVIGSCFSDNPIDSCWRGDSNWAQNRMKLADCAVGFGSSTMGGKGGDLYTVTNSDDDPVNPPGTLRYGATRDRPLWIIFSGNMNIKLKMPMYIAGYKTFDGRGAQVYIGNGGPCVFIKRVSNVIIHGLYLYGCSTSVLGNVLINESFGVEPVHPQDGDALTLRTATNIWIDHNSFSNSSDGLVDVTLTSTGVTISNNLFFNHHKVMSLGHDDAYSDDKSMKVTVAFNQFGPNCGQRMPRARYGLVHVANNNYDPWTIYAIGGSSNPTILSEGNSFTAPNESYKKQVTIRIGCKTSSSCSNWVWQSTQDVFYNGAYFVSSGKYEGGNIYTKKEAFNVENGNATPHLTQNAGVLTCS LSKRC493632 Cry j IA precursor (SEQ ID NO: 127)MDSPCLVALLVLSFVIGSCFSDNPIDSCWRGDSNWAQNRMKLADCAVGFGSSTMGGKGGDLYTVTNSDDDPVNPAPGTLRYGATRDRPLWIIFSGNMNIKLKMPMYIAGYKTFDGRGAQVYIGNGGPCVFIKRVSNVIIHGLHLYGCSTSVLGNVLINTESFGVEPVHPQDGDALTLRTATNIWIDHNSFSNSSDGLVDVTLSSTGVTISNNLFFNHHKVMLLGHDDAYSDDKSMKVTVAFNQFGPNCGQRMPRARYGLVHVANNNYDPWTIYAIGGSSNPTILSEGNSFTAPNESYKKQVTIRIGCKTSSSCSNWVWQSTQDVFYNGAYFVSSGKYEGGNIYTKKEAFNVENGNATPQLTKNAGVLT CSLSKRC1076242 Cry j II precursor - Japanese cedar (SEQ ID NO: 128)MAMKLIAPMAFLAMQLIIMAAAEDQSAQIMLDSVVEKYLRSNRSLRKVEHSRHDAINTIFNVEKYGAVGDGKHDCTEAFSTAWQAACKNPSAMLLVPGSKKFVVNNLFFNGPCQPHFTFKVDGIIAAYQNPASWKNNRIWLQFAKLTGFTLMGKGVIDGQGKQWWAGQCKWVNGREICNDRDRPTAIKFDFSTGLIIQGLKLMNSPEFHLVFGNCEGVKIIGISITAPRDSPNTDGIDIFASKNFHLQKNTIGTGDDCVAIGTGSSNIVIEDLICGPGHGISIGSLGRENSRAEVSYVHVNGAKFIDTQNGLRIKTWQGGSGMASHIIYENVEMINTSENPILINQFYCTSASACQNQRSAVQIQDVTYKNIRGTSATAAAIQLKCSDSMPCKDIKLSDISLKLTSGKIASCLNDNANGYFSGHVIPACKNLSPSAKRKESKSHKHPKTVMVENMRAYDKGNRTRILLGSRPPNCTNKCHGCSPCKAKLVIVHRIMPQEYYPQRWI CSCHGKIYHP1076241 Cry j II protein - Japanese cedar (SEQ ID NO: 129)MAMKFIAPMAFVAMQIIIMAAAEDQSAQIMLDSDIEQYLRSNRSLRKVEHSRHDAINIFNVEKYGAVGDGKHDCTEAFSTAWQAACKKPSAMLLVPGNKKFVVNNLFFNGPCQPHFTFKVDGIIAAYQNPASWKNNRIWLQFAKLTGFTLMGKGVIDGQGKQWWAGQCKWVNGREICNDRDRPTAIKFDFSTGLIIQGLKLMNSPEFHLVFGNCEGVKIIGISITAPRDSPNTDGIDIFASKNFHLQKNTIGTGDDCVAIGTGSSNIVIEDLICGPGHGISIGSLGRENSRAEVSYVHVNGAKHDTQNGLRIKTWQGGSGMASHIIYENVEMINTSENPILINQFYCTSASACQNQRSAVQIQDVTYKNIRGTSATAAAIQLKCSDSMPCKDIKLSDISLKLTSGKIASCLNDNANGYFSGHVIPACKNLSPSAKRKESKSHKHPKTVMVKNMGAYDKGNRTRILLGSRPPNCTNKCHGCSPCKAKLVIVHRIMPQEYYPQRWMCS RHGKIYHP541803 Cry j I precursor - Japanese cedar (SEQ ID NO: 130)MDSPCLVALLVLSFVIGSCFSDNPIDSCWRGDSNWAQNRMKLADCAVGFGSSTMGGKGGDLYTVTNSDDDPVNPPGTLRYGATRDRPLWIIFSGNMNIKLKMPMYIAGYKTFDGRGAQVYIGNGGPCVFIKRVSNVIIHGLHLYGCSTSVLGNVLINESFGVEPVHPQDGDALTLRTATNIWIDHNSFSNSSDGLVDVTLSSTGVTISNNLFFNHHKVMLLGHDDAYSDDKSMKVTVAFNQFGPNCGQRMPRARYGLVHVANNNYDPWTIYAIGGSSNPTILSEGNSFTAPNESYKKQVTIRIGCKTSSSCSNWVWQSTQDVFYNGAYFVSSGKYEGGNIYTKKEAFNVENGNATPQLTKNAGVLTCS LSKRC541802 Cry j I precursor - Japanese cedar (SEQ ID NO: 131)MDSPCLVALLVFSFVIGSCFSDNPIDSCWRGDSNWAQNRMKLADCAVGFGSSTMGGKGGDLYTVTNSDDDPVNPAPGTLRYGATRDRPLWIIFSGNMNIKLKMPMYIAGYKTFDGRGAQVYIGNGGPCVFIKRVSNVIIHGLYLYGCSTSVLGNVLINTESFGVEPVHPQDGDALTLRTATNIWIDHNSFSNSSDGLVDVTLTSTGVTISNNLFFNHHKVMSLGHDDAYSDDKSMKVTVAFNQFGPNCGQRMPRARYGLVHVANNNYDPWTIYAIGGSSNPTILSEGNSFTAPNESYKKQVTIRIGCKTSSSCSNWVWQSTQDVFYNGAYFVSSGKYEGGNIYTKKEAFNVENGNATPHLTQNAGVLT CSLSKRC

Dog

Canis sequences:

Can f 1 (SEQ ID NO: 132) MKTLLLTIGFSLIAILQAQDTPALGKDTVAVSGKWYLKAMTADQEVPEKPDSVTPMILKAQKGGNLEAKITMLTNGQCQNITVVLHKTSEPGKYTAYEGQRVVFIQPSPVRDHYILYCEGELHGRQIRMAKLLGRDPEQSQEALEDFREFSRAKGLNQEILELAQSETCSPGGQ Serum albumin fragment(SEQ ID NO: 133) EAYKSEIAHRYNDLGEEHFRGLVL Serum albumin fragment(SEQ ID NO: 134) LSSAKERFKCASLQKFGDRAFKAWSVARLSQRFPKADFAEISKVVTDLTKVHKECCHGDLLECADDRADLAKYMCENQDSISTKLKECCDKPVLEKSQCLAEVERDELPGDLPSLAADFVEDKEVCKNYQEAKDVFLGTFLYEYSRRHPEYSVSLLLRLAKEYEATLEKCCATDDPPTCYAKVLDEFKPLVDEPQNLVKTNCELFEKLGEYGFQNALLVRYTKKAPQVSTPTLVVEVSRKLGKVGTKCCKKPESERMSCADDFLS Can f 2 (SEQ ID NO: 135)MQLLLLTVGLALICGLQAQEGNHEEPQGGLEELSGRWHSVALASNKSDLIKPWGHFRVFIHSMSAKDGNLHGDIVPQDGQCEKVSLTAFKTATSNKFDLEYWGHNDLYLAEVDPKSYLILYMINTQYNDDTSLVAHLMVRDLSRQQDFLPAFESVCEDIGLHKDQIVVLSDDDRCQGSRD

Additional dog allergen protein (NCBI entrez accession):

1731859

Horse

Equus sequences:

1575778 Equ c1 (SEQ ID NO: 136)MKLLLLCLGLILVCAQQEENSDVAIRNFDISKISGEWYSIFLASDVKEKIEENGSMRVFVDVIRALDNSSLYAEYQTKVNGECTEFPMVFDKTEEDGVYSLNYDGYNVFRISEFENDEHIILYLVNFDKDRPFQLFEFYAREPDVSPEIKEEFVKIVQKRGIVKENIIDLTKIDRCFQLRGNGVAQA 3121755 Equ c 2 (SEQ ID NO: 137)SQXPQSETDYSQLSGEWNTIYGAASNIXK

Euroglyphus (Mite)

Euroglyphus sequences:

Eur m 1 (variant) (SEQ ID NO: 138)TYACSINSVSLPSELDLRSLRTVTPIRMQGGCGSCWAFSGVASTESAYLAYRNMSLDLAEQELVDCASQNGCHGDTIPRGIEYIQQNGVVQEHYYPYVAREQSCHRPNAQRYGLKNYCQISPPDSNKIRQALTQTHTAVAVIIGIKDLNAFRHYDGRTIMQHDNGYQPNYHAVNIVGYGNTQGVDYWIVRNSWDTTWGDNGYGYFAANINL Eur m 1 (variant) (SEQ ID NO: 139)TYACSINSVSLPSELDLRSLRTVTPIRMQGGCGSCWAFSGVASTESAYLAYRNMSLDLAEQELVDCASQNGCHGDTIPRGIEYIQQNGVVQEHYYPYVAREQSCHRPNAQRYGLKNYCQISPPDSNKIRQALTQTHTAVAVIIGIKDLNAFRHYDGRTIMQHDNGYQPNYHAVNIVGYGNTQGVDYWIVRNSWDTTWGDNGYGYFAANINL Eur m 1 (variant) (SEQ ID NO: 140)ETNACSINIGNAPAEIDLRQMRTVTPIRMQGGCGSCWAFSGVAATESAYLAYRNQSLDLAEQELVDCASQHGCHGDTIPRGIEYIQHNGVVQESYYRYVAREQSCRRPNAQRFGISNYCQIYPPNANKIREALAQTHSAIAVIIGIKDLDAFRHYDGRTIIQRDNGYQPNYHAVNIVGYSNAQGVDYWIVRNSWDTNWGDNGYGYFAANIDL Eur m 1 (variant) (SEQ ID NO: 141)ETSACRINSVNVPSELDLRSLRTVTPIRMQGGCGSCWAFSGVAATESAYLAYRNTSLDLSEQELVDCASQHGCHGDTIPRGIEYIQQNGVVEERSYPYVAREQQCRRPNSQHYGISNYCQIYPPDVKQIREALTQTHTAIAVIIGIKDLRAFQHYDGRTIIQHDNGYQPNYHAVNIVGYGSTQGVDYWIVRNSWDTTWGDSGYGYFQAGNNL

Poa (grass) sequences

113562 POLLEN ALLERGEN POA P 9 (SEQ ID NO: 142)MAVQKYTVALFLVALVVGPAASYAADLSYGAPATPAAPAAGYTPAAPAGAAPKATTDEQKMIEKINVGFKAAVAAAGGVPAANKYKTFVATFGAASNKAFAEALSTEPKGAAVDSSKAALTSKLDAAYKLAYKSAEGATPEAKYDDYVATLSEALRIIAGTLEVHGVKPAAEEVKATPAGELQVIDKVDAAFKVAATAANAAPANDKFTVFEAAFNDAIKASTGGAYQSYKFIPALEAAVKQSYAATVATAPAVKYTVFETALKKAITAMSQAQKAAKPAAAATGTATAAVGAATGAATAAAGGYKV 113561 POA P 9 (SEQ ID NO: 143)MAVHQYTVALFLAVALVAGPAASYAADVGYGAPATLATPATPAAPAAGYTPAAPAGAAPKATTDEQKLIEKINAGFKAAVAAAAGVPAVDKYKTFVATFGTASNKAFAEALSTEPKGAAAASSNAVLTSKLDAAYKLAYKSAEGATPEAKYDAYVATLSEALRIIAGTLEVHAVKPAGEEVKAIPAGELQVIDKVDAAFKVAATAANAAPANDKFTVFEAAFNDAIKASTGGAYQSYKFIPALEAAVKQSYAATVATAPAVKYTVFETALKKAITAMSQAQKAAKPAAAVTATATGAVGAATGAVGAATGAATAAAGGYKTG AATPTAGGYKV113560 POA P 9 (SEQ ID NO: 144)MDKANGAYKTALKAASAVAPAEKFPVFQATFDKNLKEGLSGPDAVGFAKKLDAFIQTSYLSTKAAEPKEKFDLFVLSLTEVLRFMAGAVKAPPASKFPAKPAPKVAAYTPAAPAGAAPKATTDEQKLIEKINTVGFKAAVAAAAGVPAASKYKTFVATFGAASNKAFAEALSTEPKGAAVASSKAVLTSKLDAAYKLAYKSAEGATPEAKYDAYVATLSEALRIIAGTLEVHGVKPAAEEVKAIPAGELQVIDKVDAAFKVAATAANAAPANDKFTVFEAAFNDAIKASTGGAYQSYKFIPALEAAVKQSYAATVATAPAVKYTVFETALKKAITAMSQAQKAAKPAAAVTGTATSAVGAATGAATAA AGGYKV

Cockroach sequences

2833325 Cr p1 (SEQ ID NO: 145)MKTALVFAAVVAFVAARFPDHKDYKQLADKQFLAKQRDVLRLFHRVHQHNILNDQVEVGIPMTSKQTSATTVPPSGEAVHGVLQEGHARPRGEPFSVNYEKHREQAIMLYDLLYFANDYDTFYKTACWARDRVNEGMFMYSFSIAVFHRDDMQGVMLPPPYEVYPYLFVDHDVIHMAQKYWMKNAGSGEHHSHVIPVNFTLRTQDHLLAYFTSDVNLNAFNTYYRYYYPSWYNTTLYGHNIDRRGEQFYYTYKQIYARYFLERLSNDLPDVYPFYYSKPVKSAYNPNLRYHNGEEMPVRPSNMYVTNFDLYYIADIKNYEKRVEDAIDFGYAFDEHMKPHSLYHDVHGMEYLADMIEGNMDSPNFYFYGSIYHMYHSMIGHIVDPYHKMGLAPSLEHPETVLRDPVFYQLWKRVDHLFQKYKNRLPRYTHDELAFEGVKVENVDVGKLYTYFEQYDMSLDMAVYVNNVDQISNVDVQLAVRLNHKPFTYNIEVSSDKAQDVYVAVFLGPKYDYLGREYDLNDRRHYFVEMDRFPYHVGAGKTVIERNSHDSNIIAPERDSYRTFYKKVQEAYEGKSQYYVDKGHNYCGYPENLLIPKGKKGGQAYTFYVIVTPYVKQDEHDFEPYNYKAFSYCGVGSERKYPDNKPLGYPFDRKIYSNDFYTPNMYFKDVIIFHKKYDEVGVQGH 2231297 Cr p2 (SEQ ID NO: 146)METHSIIGLPPFVPPSRRHARRGVGINGLIDDVIAILPVDELKALFQEKLETSPDFKALYDAIRSPEFQSIISTLNAMQRSEHHQNLRDKGVDVDHFIQLIRALFGLSRAARNLQDDLNDFLHSLEPISPRHRHGLPRQRRRSARVSAYLHADDFHKIITTIEALPEFANFYNFLKEHGLDVVDYINEIHSIIGLPPFVPPSRRHARRGVGINGLIDDVIAILPVDELKALFQEKLETSPDFKALYDAIRSPEFQSIISTLNAMPEYQELLQNLRDKGVDVDHFIRVDQGTLRTLSSGQRNLQDDLNDFLALIPTDQILAIAMDYLANDAEVQELVAYLQSDDFHKIITTIEALPEFANFYNFLKEHGLDVVDYINTEIHSIIGLPPFVPPSQRHARRGVGINGLIDDVIAILPVDELKALFQEKLETSPDFKALYDAIDLRSSRA 1703445 Bla g 2 (SEQ ID NO: 147)MIGLKLVTVLFAVATITHAAELQRVPLYKLVHVFINTQYAGITKIGNQNFLTVFDSTSCNVVVASQECVGGACVCPNLQKYEKLKPKYISDGNVQVKFFDTGSAVGRGIEDSLTISNLTTSQQDIVLADELSQEVOLSADVVVGIAAPGCPNALKGKTVLENFVEENLIAPVFSIHHARFQDGEHFGEIIFGGSDWKYVDGEFTYVPLVGDDSWKFRLDGVKIGDTTVAPAGTQAIIDTSKAIIVGPKAYVNPINTEAIGCVVEKTTTRRICKLDCSKIPSLPDVTFVINTGRNFNISSQYYIQQNGNLCYSGFQPCGHSDHFFIGDFFVDHYYSEFNWENKTMGFGRSVESV 1705483 Bla g 4 (SEQ ID NO: 148)AVLALCATDTLANEDCFRHESLVPNLDYERFRGSWIIAAGTSEALTQYKCWIDRFSYDDALVSKYTDSQGKNRTTIRGRTKFEGNKFTIDYNDKGKAFSAPYSVLATDYENYAIVEGCPAAANGHVIYVQIRFSVRRFHPKLGDKEMIQHYTLDQVNQHKKAIEEDLKHFNLKYEDLHSTCH 2326190 Bla g 5(SEQ ID NO: 149) YKLTYCPVKALGEPIRFLLSYGEKDFEDYRFQEGDWPNLKPSMPFGKTPVLEIDGKQTHQSVAISRYLGKQFGLSGKDDWENLEIDMIVDTISDFRAAIANYHYDADENSKQKKWDPLKKETIPYYTKKFDEVVKANGGYLAAGKLTWADFYFVAILDYLNHMAKEDLVANQPNLKALREKVLG LPAIKAWVAKRPPTDL

Additional cockroach sequences (NCBI Entrez accession numbers):

2580504; 1580797; 1580794; 1362590; 544619; 544618; 1531531; 1580792;1166573; 1176397; 2317849.

Allergen (general) sequences:

NCBI accession numbers

2739154; 3719257; 3703107; 3687326; 3643813; 3087805; 1864024; 1493836;1480457; 2593176; 2593174; 1575778; 763532; 746485; 163827; 163823;3080761; 163825; 3608493; 3581965; 2253610; 2231297; 2317849; 3409499;3409498; 3409497; 3409496; 3409495; 3409494; 3409493; 3409492; 3409491;3409490; 3409431; 3409488; 3409487; 3409486; 3409485; 3409484; 3409483;3409482; 3409481; 3409480; 3409479; 3409478; 3409477; 3409476; 3409475;3409474; 3409473; 3409472; 3409471; 3409470; 3409469; 3409468; 3409467;3409466; 3409465; 3409464; 3409463; 3409462; 3409461; 3409460; 3409459;3409458; 3409457; 3409456; 3318885; 3396070; 3367732; 1916805; 3337403;2851457; 2851456; 1351295; 549187; 136467; 1173367; 2499810; 2498582;2498581; 1346478; 1171009; 126608; 114091; 2506771; 1706660; 1169665;1169531; 232086; 416318; 114922; 2497701; 1703232; 1703233; 1703233;1703232; 3287877; 3122132; 3182907; 3121758; 3121756; 3121755; 3121746;3121745; 3319925; 3319923; 3319921; 3319651; 3318731; 3318779; 3309647;3309047; 3309045; 3309043; 3309041; 3309039; 3288200; 3288068; 2924494;3256212; 3256210; 3243234; 3210053; 3210052; 3210051; 3210050; 3210049;3210048; 3210047; 3210046; 3210045; 3210044; 3210043; 3210042; 3210041;3210040; 3210039; 3210038; 3210037; 3210036; 3210035; 3210034; 3210033;3210032; 3210031; 3210030; 3210029; 3210028; 3210027; 3210026; 3210025;3210024; 3210023; 3210022; 3210021; 3210020; 3210019; 3210018; 3210017;3210016; 3210015; 3210014; 3210013; 3210012; 3210011; 3210010; 3210009;3210008; 3210007; 3210006; 3210005; 3210004; 3210003; 3210002; 3210001;3210000; 3209999; 3201547; 2781152; 2392605; 2392604; 2781014; 1942360;2554672; 2392209; 3114481; 3114480; 2981657; 3183706; 3152922; 3135503;3135501; 3135499; 3135497; 2414158; 1321733; 1321731; 1321728; 1321726;1321724; 1321722; 1321720; 1321718; 1321716; 1321714; 1321712; 3095075;3062795; 3062793; 3062791; 2266625; 2266623; 2182106; 3044216; 2154736;3021324; 3004467; 3005841; 3005839; 3004485; 3004473; 3004471; 3004469;3004465; 2440053; 1805730; 2970629; 2959318; 2935527; 2935416; 809536;730091; 585279; 584968; 2498195; 2833325; 2498604; 2498317; 2498299;2493414; 2498586; 2498585; 2498576; 2497749; 2493446; 2493445; 1513216;729944; 2498099; 548449; 465054; 465053; 465052; 548671; 548670; 548660;548658; 548657; 2832430; 232084; 2500822; 2498118; 2498119; 2498119;2498118; 1708296; 1708793; 416607; 416608; 416608; 416607; 2499791;2498580; 2498579; 2498578; 2498577; 2497750; 1705483; 1703445; 1709542;1709545; 1710531; 1352699; 1346568; 1346323; 1346322; 2507248; 11352240;1352239; 1352237; 1352229; 1351935; 1350779; 1346806; 1346804; 1346803;1170095; 1168701; 1352506; 1171011; 1171008; 1171005; 1171004; 1171002;1171001; 1168710; 1168709; 1168708; 1168707; 1168706; 1168705; 1168704;1168703; 1168702; 1168696; 1168391; 1168390; 1168348; 1173075; 1173074;1173071; 1169290; 1163170; 1168402; 729764; 729320; 729979; 729970;729315; 730050; 730049; 730048; 549194; 549193; 549192; 549191; 549190;549131; 549188; 549185; 549184; 549183; 549182; 549181; 549180; 549179;464471; 585290; 416731; 1169666; 113478; 113479; 113477; 113476; 113475;130975; 119656; 113562; 113561; 113560; 416610; 126387; 126386; 126385;132270; 416611; 416612; 416612; 416611; 730035; 127205; 1352238; 125887;549186; 137395; 730036; 133174; 114090; 131112; 126949; 129293; 124757;129501; 416636; 2801531; 2796177; 2796175; 2677826; 2735118; 2735116;2735114; 2735112; 2735110; 2735108; 2735106; 273531; 2735102; 2735100;2735098; 2735096; 2707295; 2154730; 2154728; 1684720; 2580504; 2465137;2465135; 2465133; 2465131; 2465129; 2465127; 2564228; 2564226; 2564224;2564222; 2564220; 2051993; 1313972; 1313970; 1313968; 1313966; 2443824;2488684; 2488683; 2488682; 2488681; 2488680; 2488679; 2488678; 2326190;2464905; 2415702; 2415700; 2415698; 2398759; 2398757; 2353266; 2338288;1167836; 414703; 2276458; 1684718; 2293571; 1580797; 1580794; 2245508;2245060; 1261972; 2190552; 1881574; 511953; 1532058; 1532056; 1532054;1359436; 666007; 487661; 217308; 1731859; 217306; 217304; 1545803;1514943; 577696; 516728; 506858; 493634; 493632; 2154734; 2154732;543659; 1086046; 1086045; 2147643; 2147642; 1086003; 1086002; 1086001;543675; 543623; 543509; 543491; 1364099; 2147108; 2147107; 1364001;1085628; 631913; 631912; 631911; 2147092; 477301; 543482; 345521;542131; 542130; 542129; 100636; 2146809; 480443; 2114497; 2144915;72355; 71728; 319828; 1082946; 1082945; 1082944; 539716; 539715; 423193;423192; 423191; 423190; 1079187; 627190; 627131; 627188; 627187; 482382;1362656; 627186; 627185; 627182; 482381; 85299; 85298; 2133756; 2133755;1079186; 627181; 32314; 32313; 112559; 112558; 1362590; 2133564;1085122; 1073171; 627144; 627143; 627142; 627141; 280576; 102835;102834; 102833; 102832; 84703; 84702; 84700; 84699; 84698; 84696;477888; 477505; 102575; 102572; 478272; 2130094; 629813; 629812; 542172;542168; 542167; 481432; 320620; 280414; 626029; 542132; 320615; 320614;100638; 100637; 100635; 82449; 320611; 320610; 280409; 320607; 320606;539051; 539050; 539049; 539048; 322803; 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1829319; 1829318; 1829317; 1829316; 1829315; 1829314; 1825459;1803187; 159653; 1773369; 1769849; 1769847; 608690; 310877; 310875;1438761; 1311513; 1311512; 1311511; 1311510; 1311509; 1311631; 1246120;1246119; 1246118; 1246117; 1246116; 1478293; 1478292; 1311642; 1174278;1174276; 1086972; 1086974; 1086976; 1086978; 1086978; 1086976; 1086974;1086972; 999009; 999356; 999355; 994866; 994865; 913758; 913757; 913756;913285; 913283; 926885; 807138; 632782; 601807; 546852; 633938; 544619;544618; 453094; 451275; 451274; 407610; 407609; 404371; 409328; 299551;299550; 264742; 261407; 255657; 250902; 250525; 1613674; 1613673;1613672; 1613671; 1613670; 1613304; 1613303; 1613302; 1613240; 1613239;1613238; 1612181; 1612180; 1612179; 1612178; 1612177; 1612176; 1612175;1612174; 1612173; 1612172; 1612171; 1612170; 1612169; 1612168; 1612167;1612166; 1612165; 1612164; 1612163; 1612162; 1612161; 1612160; 1612159;1612158; 1612157; 1612156; 1612155; 1612154; 1612153; 1612152; 1612151;1612150; 1612149; 1612148; 1612147; 1612146; 1612145; 1612144; 1612143;1612142; 1612141; 1612140; 1612139; 1093120; 447712; 447711; 447710;1587177; 158542; 1582223; 1582222; 1531531; 1580792; 886215; 1545317;1545315; 1545313; 1545311; 1545831; 1545887; 1545885; 1545883; 1545881;1545879; 1545877; 1545875; 166486; 1498496; 1460058; 972513; 1009442;1009440; 1009438; 1009436; 1009434; 7413; 1421808; 551228; 452606;32905; 1377859; 1364213; 1364212; 395407; 22690; 22688; 22686; 22684;488605; 17680; 1052817; 1008445; 1008443; 992612; 706811; 886683;747852; 939932; 19003; 1247377; 1247375; 1247373; 862307; 312284;999462; 999460; 999458; 587450; 763064; 886209; 1176397; 1173557;902012; 997915; 997914; 997913; 997912; 997911; 997910; 99790; 997908;997907; 997906; 997905; 997904; 997903; 997902; 997901; 997900; 997319;997318; 997317; 997316; 997315; 997314; 997313; 997312; 910984; 910983;910982; 910981; 511604; 169631; 169629; 169627; 168316; 168314; 607633;555616; 293902; 485371; 455288; 166447; 166445; 166443; 166435; 162551;160780; 552080; 156719; 156715; 515957; 515956; 515955; 515954; 515953;459163; 166953; 386678; 169865.

Particularly preferred allergens/antigens include: cat dander proteinFel d1 ; Ragweed proteins Der P1, Der P2 and Der P7; Ragweed protein amba 1.1, a 1.2, al.3 or al.4; Rye grass proteins lol pl and lol p5;Timothy grass proteins phl pl and phl p5; Bermuda grass protein Cyn d 5;Alternaria alternate proteins Alt a 1, Alt a 2 and Enolase (Alt a 6);Birch protein Bet v1 and P14; German Cockroach proteins Bla g 1, Bla g2, Bla g 3, Bla g 4, Bla g 5 and Bla g 6; Mugwort protein Art v 1;Russian thistle protein Sal k 1 and Sal k 2; peanut Ara h1 , Ara h2, Arah3, Ara h4, Ara h5, Ara h6, plant profilins or lipid transfer proteinsor a human leukocyte antigen.

Delivery Methods

Once formulated the compositions of the invention can be delivered to asubject in vivo using a variety of known routes and techniques. Forexample, a composition can be provided as an injectable solution,suspension or emulsion and administered via parenteral, subcutaneous,epicutaneous, epidermal, intradermal, intramuscular, intraarterial,intraperitoneal, intravenous injection using a conventional needle andsyringe, or using a liquid jet injection system. Compositions can alsobe administered topically to skin or mucosal tissue, such as nasally,intratracheally, intestinal, rectally or vaginally, or provided as afinely divided spray suitable for respiratory or pulmonaryadministration. Other modes of administration include oraladministration, suppositories, sublingual administration, and active orpassive transdermal delivery techniques.

Where a peptide of the invention is to be administered, it is preferredto administer the peptide to a site in the body where it will have theability to contact suitable antigen presenting cells, and where it, orthey, will have the opportunity to contact T cells of the individual.Where an APC is to be administered, it is preferred to administer theAPC to a site in the body where it will have the ability to contact, andactivate, suitable T cells of the individual.

Delivery Regimes

Administration of the peptides/polynucleotides/cells (such as thecomposition containing a plurality of peptides) may be by any suitablemethod as described above. Suitable amounts of the peptide may bedetermined empirically, but typically are in the range given below. Asingle administration of each peptide may be sufficient to have abeneficial effect for the patient, but it will be appreciated that itmay be beneficial if the peptide is administered more than once, inwhich case typical administration regimes may be, for example, once ortwice a week for 2-4 weeks every 6 months, or once a day for a weekevery four to six months. As will be appreciated, each peptide orpolynucleotide, or combination of peptides and/or polynucleotides may beadministered to a patient singly or in combination.

Dosages for administration will depend upon a number of factorsincluding the nature of the composition, the route of administration andthe schedule and timing of the administration regime. Suitable doses ofa molecule of the invention may be in the order of up to 15 μg, up to 20μg, up to 25 μg, up to 30μg, up to 50 μg, up to 100 μg, up to 500 μg ormore per administration. Suitable doses may be less than 15 μg, but atleast ing, or at least 2 ng, or at least 5 ng, or at least 5 ng, orleast 100 ng, or at least 500 ng, or at least lm, or at least 10 μg. Forsome molecules of the invention, the dose used may be higher, forexample, up to 1 μg, up to 2 μg, up to 3 μg, up to 4 μg, up to 5 μg orhigher. Such doses may be provided in a liquid formulation, at aconcentration suitable to allow an appropriate volume for administrationby the selected route.

Kits

The invention also relates to a combination of components describedherein suitable for use in a treatment of the invention which arepackaged in the form of a kit in a container. Such kits may comprise aseries of components to allow for a treatment of the invention. Forexample, a kit may comprise one or more different peptides,polynucleotides and/or cells of the invention, or one or more peptides,polynucleotides or cells of the invention and one or more additionaltherapeutic agents suitable for simultaneous administration, or forsequential or separate administration. The kit may optionally containother suitable reagent(s) or instructions and the like.

The invention is illustrated by the following Examples:

EXAMPLE 1 Potential T Cell Epitopes

The regions of Amb a 1 described below were identified as potentiallycomprising one or more T cell epitopes:

TABLE 2 RESIDUES IN REGION OF AMB A 1 INTEREST (isoform 1.3) SEQUENCE A178-189 GMIKSNDGPPIL (SEQ ID NO: 32) B 202-213GSSQIWIDHCSLSKS (SEQ ID NO: 5) C 343-354 DKDLLENGAIFVTSGSDPVLTPVQ (SEQ ID NO: 33) D 364-375 PVQSAGMIPAEPGEA (SEQ ID NO: 34) E 103-114EGTLRFAAAQNRPLW (SEQ ID NO: 35) F 130-141 QELVVNSDKTIDGRGVKVEII (SEQ ID NO: 36) G 376-387 GEAAIKLTSSAGVFSCHP (SEQ ID NO: 37) H 226-237GSTHVTISNCKF (SEQ ID NO: 22) I 280-297FOFFQVVNNNYDRWOTYA (SEQ ID NO: 38) J  38-48 ETRSLQACEALN (SEQ ID NO: 39)

The sequences provided for regions G, I and J in the priorityapplication, GB 0815258.3 are as follows: GEAAIKLTSSAGVLSCRP(Region G,SEQ ID NO: 40), HGFFQVVNNNYDRGTYA (Region I, SEQ ID NO: 41) andETRRLTTSGAYN (Region J, SEQ ID NO: 42). These sequences have beencorrected in Table 2 such that they correspond to the relevant regionsfrom Amb a 1 isoform 1.3 full length sequence as provided below.

The regions shown in Table 2 were then further analysed to find which ofthem were highly conserved between the 4 different Amb al isoforms asshown below (1.1, 1.2, 1.3 and 1.4). For the below sequences, thefollowing text styles are used to indicate regions of interest: RegionA, Region B, Region C, Region D, Region E, Region G, Region H, Region I,Region J

Amb 1.1 (SEQ ID NO: 43)

Amb 1.2 (SEQ ID NO: 44)

Amb 1.3 (SEQ ID NO: 45)

Amb 1.4 (SEQ ID NO: 46)

The following peptides were considered to be highly conserved andtherefore taken forward for further testing:

TABLE 3 Derived from region SEQ ID Peptide of interest Sequence NO:RGW01 A GMIKSNDGPPI  1 RGW01A A GLIKSHDGPPV  2 RGW01B A GLIKSNDGPAA  3RGW02 B GSSQIWIDHSSLSKS  4 RGW02A B GSSQIWIDHCSLSKS  5 RGW02B BGGSQIWIDHCSLSKA  6 RGW03 C KDLLENGAIFVTSG  7 RGW03A C DVFENGAIFVPSG  8RGW03B C RDLLENGAIFLPSG  9 RGW04 D KAGMIPAEPGEA 10 RGW4A D SAGMIPAEPGEA11 RGW05 E KEGTLRFAAAQNRP 12 RGW05A E KEGTLRFGAAQNRP 13 RGW06 FVVNSDKTIDGRGVKVE 14 RGW06A F AINNDKTIDGRGAKVE 15 RGW07 G GEAAIKLTSSAGVLS16 RGW07A G GEAVLRLTSSAGVLS 17 RGW07B G GESALSLTSSAGVLS 18 RGW07C GKGEAAIKLTSSAGVLSK 19 RGW07D G KGEAAIKLTSSAGVLSKK 20 RGW08 H GSTHVTISNSKF21 RGW08A H GSTHVTISNCKF 22 RGW08B H GSTHFTVSNCLF 23 RGW08C HGSTHFTVSNSLF 24 RGW08D H GTTRLTVSNSLF 25 RGW09 J ETRRSLKTSGAYN 26 RGW10I FGFFQVVNNNYD 27 RGW10A I HGFFQVVNNNYD 28 RGW11 I VNNNYDRWGTYA 29RGW11A I VNNNYDKWGSYA 30 RGW11B I VNNNYERWGSYA 31

As will be apparent, the sequences above are not necessarily identicalto the native sequences of the regions of interest. In particular, thepeptides of the invention may be engineered to improve solubility and/orreduce dimer formation and/or reduce the likelihood of IgE cross-linkingrelative to the native sequences. The following table

(Table 4) provides specific illustrations of the above principles asapplied by the inventors to produce the peptides of Table 3 (SEQ ID NOS:1 to 31).

TABLE 4 Sequence Identificat  ^(n) pI GRAVY Comments GMIKSNDGPPIL Region A 5.84 -0.083 (SEQ ID NO: 32) GMIKSNDGPPI RGW01 5.84 -0.436Engineered peptide has (SEQ ID: 1) lower GRAVY score: more solubleGSSQIWIDHCSLSKS  Region B 6.73 -0.273 (SEQ ID No: 5) GSSQIWIDHSSLSKSRGW02 6.73 -0.493 Engineered peptide has ser (SEQ ID: 4)in place of cys (avoids dimer formation) and has lower GRAVY score: moresoluble DKDLLENGAIFVTSGSDPVL Region C TPVQ (SEQ ID NO: 33)RDLLENGAIFLPSG RGW03B Engineered peptide (SEQ ID: 9)significantly shorter: avoids risk of histamine release viacross linking IgE AIKLTSSAGVFSCHP  Region G (SEQ ID NO: 37)GEAAIKLTSSAGVLS RGW07 6.00  0.69 Engineered peptide lacks (SEQ ID: 16)cys in last three residues (to prevent dimer formation)and has L replacing F as occurs in 1.1, 1.2 and 1.4

Rows highlighted in grey in the above table represent the nativesequence of a region. The peptides modified according to the inventionare shown beneath each native sequence. Residues in bold and underlinedrepresent additions to or substitutions of the native sequence.

Cytokine Assays and Selection of Preferred Combinations

Cytokine secretion profiles from PBMC's of 50 individuals were analysedin response to the peptide stimulation using the peptides of SEQ ID NOS1 to 31. Supernatants from the cytokine release assay were tested forthe presence of IL-13 and IFN-gamma, using a multiplex bead array assay.

A typical cytokine release assay requires 40×10⁶ PBMC's per subject. Inmore detail, 250 μl of a 200 μg/ml solution of the appropriate antigenor peptide concentration is distributed into the appropriate wells of 48well plates. Plates are the incubated in a humidified 5% CO2 incubatorat 37° C. for a maximum of 4 hours. 250 μl of a 5×10⁶ cell/ml PBMCsuspension is then added to each well and the plates returned to theincubator for 5 days. Following stimulation, samples of culturesupernatant are harvested for testing by multiplex bead assay accordingto standard protocols. The data was analysed for subjects having aresponse to Interferon gamma or IL-13>100 pg/ml and the peptides wereprioritised on the basis of the % responder rate as shown below in Table5.

TABLE 5 Number of responders >100 pg/ml % responders RGW 01 34 68 RGW01A 25 50 RGW 03 25 50 RGW 01B 24 48 RGW 04 23 46 RGW 03A 22 44 RGW 03B22 44 RGW 10 21 42 RGW 04A 20 40 RGW 10A 20 40 RGW 05 19 38 RGW 02 18 36RGW 09 18 36 RGW 06 17 34 RGW 06A 17 34 RGW 11A 17 34 RGW 05A 16 32 RGW08B 10 20 RGW 11B 10 20 RGW 02B 9 18 RGW 07A 9 18 RGW 11 8 16 RGW 08A 612 RGW 08D 6 12 RGW 02A 5 10 RGW 07B 5 10 RGW 08 4 8 RGW 07 2 4 RGW 08C2 4

Based on the above a preferred combination of the invention was selectedto contain: RGW01 (potentially substituted with RGW01A or RGW01B); andone of RGW03, RGW03A or RGW03B; and one of RGW04 or RGW04A.

From the remaining peptides, the next most potent are RGW02, RGW09,RGW06, RGW06A, RGW10 or RGW10A, RGW05 or RGW05A. A preferred peptidecombination may typically comprise at least one additional peptideselected from this group.

Since solubility is a key criterion for peptides to be administered topatients, further in vitro solubility testing was performed to evaluatethe solubility of peptides in an acidic environment (pH 2.97 0.1mM HCl,0.5% (w/v), 1-thioglycerol (ca. 46 mM), 230 mM trehalose). For example,RGW03B was found to have a solubility of 3.85 mg/ml compared to <0.62mg/ml for RGW03. Accordingly, the inventors have determined that RGW03Bis preferred over RGW03.

FIG. 1 shows the number of individuals who respond to a core mixture ofRGW01, RGW03B and RGW04A. A combined analysis of the positive IFNgammaor IL-13 responses for the peptides represented in the core mixtureshows that these 3 peptides provide coverage for 38/50 ragweed allergicindividuals (76% of the study population). This indicates that thepeptides of the core mixture bind to most MHC class II DR molecules andthat these complexes are recognized by T cells in the majority ofallergic individuals. The incremental effect of adding RGW02, RGW05 andRGW06A is also shown. The benefit of adding epitopes from the secondgroup of peptides is clearly shown.

A further cytokine assay was performed to evaluate IL-10 release fromthe same panel of individuals, induced by the peptides of SEQ ID NOS. 1to 31. Analysis of IL-10 response shows that RGW07 and RGW07B inducesignificant quantities of IL-10 (FIG. 2). RGW07 on its own induced IL-10in 49/50 individuals. A preferred peptide combination may thereforetypically comprise at least one additional peptide selected from theRGW07 group. A combined analysis of the positive IFNgamma or IL-13responses for the peptides represented in the mixture of 6 peptidesidentified in FIG. 1 (RGW01+RGW03B+RGW04A+RGW02+RGW05+RGW06A) togetherwith RGW07 gave a positive IFNgamma or IL-13 response in 49/50 subjects.Thus, this peptide combination effectively covers the whole studypopulation, and complete coverage would be expected on use of improvedRGW07 solubility variants (see below). Peptide combinations comprisingRGW01+RGW03B+RGW04A+RGW02+RGW05+RGW06A) or variants thereof togetherwith RGW07 or variants thereof also have the advantage of providing fora positive IL-10 response (see below and FIG. 3).

Solubility testing showed that RGW07 had poor solubility in an acidicenvironment and so additional lysine residues were added to RGW07(indicated in Table 6). The modified peptides were tested for solubilityand their ability to induce IL-10 from PBMC in the presence of themixture of peptides identified in FIG. 1(RGW01+RGW03B+RGW04A+RGW02+RGW05+RGW06A). Controls included the peptidemixture with no RGW07 variant and with whole Amb a 1 allergen. Theresults are shown in FIG. 3.

TABLE 6 Solubility of RGW07 and variants Residues Isoele- in ctricSolubility Peptide Sequence Amb a 1 point (pI) GRAVY mg/ml RGW07GEAAIKITSSAGVLS 376-390  6.00  0.69 0.7  (SEQ ID NO: 16) RGW07CKGEAAIKLTSSAGVLSK K376-  9.70  0.15 1.55 (SEQ ID NO: 19) 390K RGW07DKGEAAIKLTSSAGVLSKK 1076- 10.00 -0.07 4.26 (SEQ ID NO: 20) 390KK

Based on these analyses, RGW07D was found to be preferred both forsolubility and for its ability to induce IL-10 in the presence of theother six peptides.

EXAMPLE 2 Histamine Release Assay

The purpose of this assay was to identify individual peptides that arecapable of activating blood basophils (as a surrogate for tissue mastcells) resulting in histamine release that may result in allergicreactions during therapy. Peptides or combinations of peptides thatinduce histamine release frequently may be considered unsuitable forinclusion in the peptide vaccine.

Histamine release requires the crosslinking of adjacent specific IgEmolecules on the surface of the basophil. The peptides being evaluatedwere small (11 to 18 amino acids in length) and should not, therefore,possess significant tertiary structure that would enable them to retainthe conformation of an IgE-binding epitope of the whole molecule.Furthermore, peptide monomers in solution, even if they are bound byIgE, should not be able to cros slink adjacent IgE molecules.

Histamine release from fresh peripheral whole blood from ragweedallergic subjects was evaluated. Peripheral blood basophils were used asa surrogate for tissue mast cells which were not practical to assay.Blood was incubated in vitro with individual peptides identified inExample 1 (RGW01, RGW02, RGW03B, RGW04A, RGW05, RGW06A, RGW07 ANDRGW07D). Additionally, responses to preferred mixtures of 7 peptidesidentified in Example 1 were analysed. The tested preferred mixtures of7 peptides consisted of i) RGW01, RGW02, RGW03B, RGW04A, RGW05, RGW06A,RGW07 and ii) RGW01, RGW02, RGW03B,

RGW04A, RGW05, RGW06A, and RGW07D.

Histamine release in response to whole ragweed allergen extract wasmeasured in each subject to confirm basophil sensitisation. A positivecontrol, representing total histamine release, generated byfreeze/thawing the cells twice, was included in each assay. A negativecontrol for spontaneous histamine release was generated by incubatingcells in buffer only.

The assay was performed using the Immunotech Histamine ReleaseImmunoassay kit according to the manufacturer's instructions. Followingthe in vitro challenge of blood basophils with peptides, peptide mixes,whole allergen or buffer in microtitre plate wells, supernatants wereremoved and the histamine in the samples converted to acyl histamine.Acylated samples were tested by a competitive acyl histamine ELISA.

Peptides were assayed for their ability to induce histamine release overa 5 log 10 range (1 to 10,000 ng/ml). The concentration range assayedwas selected based on theoretical in vivo doses of peptide that may beachieved during therapy. For example, a 31 μg dose (approximately 3nmol/peptide equivalent) of each peptide entering a blood volume of 5litres, would result in a blood concentration of 6ng/ml, at the lowerend of the histamine release assay dose range. Whole ragweed allergenextract was used over a slightly higher concentration range (10 to100,000 ng/ml).

Single measurements were performed for each dilution. After completionof the ELISA, individual histamine levels were determined byinterpolation from the standard curve generated in the ELISA assay.Results from samples were adjusted to allow for dilution. Where two ormore consecutive dilutions of a peptide/allergen preparationelicited >15% of the total histamine release seen in the freeze thawedpositive control (>15% of positive control), or where a single valueof >15% of positive control was achieved at the highest concentrationtested (10 μg/mL for peptides), this was considered a “positivehistamine release”.

A total of 49 histamine release assays were completed during the study.Of these 6 assays were rejected, due to unacceptably high levels (>15%of positive control) of spontaneous release in the medium plus buffernegative control wells. Therefore a total of 43 subjects were includedin the analysis. 20 of these subjects were tested with the RGW07Dpeptide and the mix containing it. The study findings are summarised inTable 7.

TABLE 7 Subjects with Mean histamine Maximum histamine release of 43 or20 histamine release >15% subjects as a % of release (% of Peptide or ofpositive positive control positive control combination control at 10μg/mL at 10 μg/mL) 1) RGW 01 0/43 1% 0% 2) RGW 03B 0/43 1% 0% 3) RGW 04A0/43 0% 0% 4) RGW 05 0/43 0% 0% 5) RGW 02 0/43 1% 0% 6) RGW 06A 0/43 1%0% 7) RGW 07 0/43 1% 0% 8) Peptides 1-6 0/43 1% 0% and RGW07 incombination 9) RGW 07D 0/20 3% 0% 10) Peptides 1-6  2/20* 5% 20%  andRGW07D in combination Ragweed allergen 28/43  25%  78%  control*Peptides 1-6 and RGW07D in combination were >15% in two subjects onlyat 10 μg/ml.

Results are shown for the highest dose of peptides tested in the assay(10 μg/ml). The ragweed allergen control induced significant histaminerelease in 65% of the 43 subjects. Even at the lowest concentration of10 ng/ml, the whole allergen extract induced significant histaminerelease in 22/43 (51%) of individuals with a mean release for the 43subjects of 30% of positive control. The crude whole ragweed extractcontains approximately 0.5% of major allergen Amb al, emphasizing theexquisite sensitivity of the in vitro basophil assay for assessing thesafety of the peptides which at 10 ug/ml are present at >10,000 foldexcess over Amb al in the crude extract at 10 ng/ml.

The data shows that the eight individual peptides do not elicitsignificant histamine release from the basophils of ragweed allergicindividuals when compared to whole allergen. A combination of peptidesRGW01, RGW02, RGW03B, RGW04A, RGW05, RGW06A, RGW07 also failed to elicitsignificant histamine release in the 43 subjects.

The combination of peptides RGW01, RGW02, RGW03B, RGW04A, RGW05, RGW06A,and RGW07D gave a weak positive response in 2 individuals with values of16% and 20% of positive control at the highest peptide concentration of10 μg/mL. The % release values at the four lower concentrations testedwere <15%, i.e. negative. Given the large excess of peptide dose testedin this assay compared to likely blood concentrations of the peptidesfollowing clinical dosing—the highest concentration testedrepresents >1000-fold excess over the expected blood concentrations—itis not anticipated that this peptide combination will cause significanthistamine release either by IgE-mediated or direct peptide-mediatedbasophil or mast cell activation and degranulation.

EXAMPLE 3 Clinical Trial of Peptide Combination

A preferred mixture of 7 peptides consisting of peptides RGW01, RGW02,RGW03B, RGW04A, RGW05, RGW06A, and RGW07D is tested in a randomised,placebo-controlled, blind clinical trial. The efficacy of this mixturein reducing allergic symptoms is evaluated. The study design of theclinical trial is in accordance with good clinical practice guidelines.

Ragweed-allergic subjects are screened to identify late phase skinresponse (LPSR) and conjunctival provocation test (CPT) scores followingchallenge with ragweed allergen. Details of LPSR and CPT assays areprovided below. Titrations are performed in order to identify theminimally effective concentrations of whole allergen for generation ofan appropriate LPSR and CPT score. Blood samples are taken to evaluatelevels of ragweed-specific IgE.

Baseline skin and conjunctival responses to ragweed allergen for allsubjects are established using a Baseline Challenge which takes placebetween 1 to 4 weeks prior to study medication administration. Anintradermal injection of ragweed allergen at the minimally effectiveconcentration identified in screening is administered into the volarsurface of a selected forearm. Subjects are assessed to ensure that theyexperience a early phase skin response (EPSR), a CPT and a Late-PhaseSkin Response (LPSR) to whole ragweed allergen, and the magnitude of thebaseline reaction is recorded as follows:

15 minutes (±3 minutes) after the injection, the outline of any EPSR isdrawn onto the skin with a ballpoint pen. The longest and orthogonaldiameters are measured and recorded for each response, and the area ofthe response in each arm is calculated.

At a minimum of 30 minutes after the injection, ragweed allergen at theminimally effective concentration identified in screening is instilledinto a selected eye. Subject rated itching and observer rated rednessand watering is then scored after 5 minutes (±2 minutes). The scoringsystem is shown in Table 8 below.

Eight hours (±10 minutes) after each injection the outline of anylate-phase response is drawn onto the skin with a ballpoint pen. Thelongest and orthogonal diameters are measured and recorded for eachresponse, and the area of the response in each arm is calculated.

Subjects who produce a suitable baseline reaction are assigned to dosinggroups, randomised and entered into the Treatment Phase.

TABLE 8 Symptom Score Redness of conjunctiva 0 = none (observer rated) 1= slight (just perceptible) 2 = moderate (noticeable redness) 3 = severe(intense angry redness) Watering (observer rated) 0 = none 1 = slight(just perceptible) 2 = moderate (occasional tearing) 3 = severe (tearsrunning down cheek) Itching (self-evaluated) 0 = none 1 = slight(occasional tingling sensation) 2 = moderate (noticeable itching but noneed to rub eye) 3 = severe (itching with need to rub eye) 4 =unbearable (unbearable itching with a compulsive desire to rub eye)

The Treatment Phase consists of a period of 6 weeks for each subject.During this period one group of subjects receive a single intradermalinjection of either the preferred mixture (0.03, 0.3, 3, 1, 12 nmol ofeach peptide per dose) or diluent placebo at Treatment Phase Visit 1 onday one. Intradermal injections are made into the flexor surface of theselected forearm. A repeat administration is then performed at TreatmentPhase Visits 2, 3 and 4, each two weeks apart (14±2 days). A cohort of10 subjects receives treatment at each dose level (8 receive thepreferred mixture and 2 placebo). The first cohort receives 0.03 nmol ofeach peptide in the mixture and each subsequent cohort in the groupreceives the next higher dose level.

19 to 28 weeks after the beginning of treatment subjects have their skinand conjunctival responses to whole allergen retested in apost-treatment challenge (PTC). Skin responses to ragweed allergen areassessed by measurement of the EPSR and LPSR as described above. Theaverage area of response is calculated as described above. Conjunctivalresponses to ragweed allergen are assessed by measurement of the CPT asdescribed above. Blood samples are taken for measurement of ragweedspecific IgE.

The average EPSR area after treatment is compared to the baseline EPSRarea for each subject. The overall change in EPSR area for all tenpatients in each cohort is then evaluated. The average LPSR area aftertreatment is compared to the baseline LPSR area for each subject. Theoverall change in LPSR area for all ten patients in each cohort is thenevaluated. The CPT score after treatment is compared to the baseline CPTscore for each subject. The overall change in CPT score for all tenpatients in each cohort is then evaluated.

Further Embodiments of the Invention

I. A composition for use in preventing or treating allergy to ragweed bytolerisation comprising at least three polypeptides, wherein thepolypeptides are independently selected from any of the following:

(i) a polypeptide of any of SEQ ID NO's. 1 to 31; or

(ii) a variant of a polypeptide according to (i), wherein said variantis a polypeptide of length 9 to 30 amino acids that comprises a regionconsisting of:

-   -   any of the sequences of (i); or    -   a sequence which has at least 65% homology to any of the        sequences of (i) which sequence is capable of tolerising an        individual to any of the sequences of (i), or

(iii) a variant of a polypeptide according to (i), wherein said variantis a polypeptide of length 9 to 30 amino acids that comprises a regionconsisting of a sequence that represents either:

-   -   a fragment of any of the sequences of (i); or    -   a homologue of a fragment of any of the sequences of (i),

which sequence is capable of tolerising an individual to any of thesequences of (i) and has a length of at least 9 amino acids, and whereinsaid homologue has at least 65% homology to any 9 contiguous amino acidsin any of the sequences of (i).

II. The composition according to item I, wherein the composition:

a) is capable of tolerising at least 50% or at least 60% of a panel ofragweed allergic individuals in the population; and/or

b) comprises at least three polypeptides selected from item I(i) orvariants thereof as defined in item I(ii) or I(iii), and/or

c) comprises at least one further polypeptide up to a total of thirteenunique/different polypeptides, wherein the further polypeptides:

-   -   comprise a sequence having at least 65% sequence identity to at        least 9 or more contiguous amino acids in any of SEQ ID NO: 1 to        31 above not selected in (a); and    -   are 9 to 30 amino acids in length; and/or

d) comprises up to a maximum of thirteen polypeptides.

III. The composition according to item I or II, comprising at least onepolypeptide according to item II(c) which is 9 to 20 or 13 to 17 aminoacids in length and/or wherein said polypeptide has at least 70%sequence identity to at least 9 or more contiguous amino acids in any ofSEQ ID NO: 1 to 31.

IV. A composition according to any one of the preceding items,comprising at least one polypeptide selected from a polypeptide ofRGW01, RGW01A or RGW01B (SEQ ID NOS: 1, 2 or 3) or a variant thereof asdefined in item I(ii) or (iii).

V. A composition according to item IV, wherein the at least onepolypeptide is the polypeptide RGW01, or a variant thereof.

VI. A composition according to item IV or V, comprising at least onepolypeptide selected from a polypeptide of RGW03B, RGW03A or RGW03 (SEQID NOS: 9, 8 or 7), or a variant thereof as defined in item I(ii) or(iii);

VII. A composition according to item VI wherein the at least onepolypeptide is the polypeptide RGW03B, or a variant thereof.

VIII. A composition according to any one of items IV to VII, comprisingat least one polypeptide selected from a polypeptide of RGW04 or RGW04A(SEQ ID NOS: 10 or 11), or a variant thereof as defined in item I(ii) or(iii).

IX. A composition according to any one of items IV to VIII, comprisingat least one polypeptide selected from a polypeptide of RGW02, RGW09,RGW06, RGW06A, RGW10 or RGW10A, RGW05 or RGW05A (SEQ ID NOS: 4, 26, 14,15, 27, 12 or 13), or a variant thereof as defined in item I(ii) or(iii).

X. A composition according to any one of items IV to IX, comprising atleast one polypeptide selected from a polypeptide of RGW07, RGW07C orRGW07D (SEQ ID NOS: 16, 19 or 20), or a variant thereof as defined initem I(ii) or (iii).

XI. A composition according to item X, wherein the at least onepolypeptide is RGW07D (SEQ ID NO: 20).

XII. A composition according to any one of the preceding itemsconsisting of:

a) at least one of the polypeptides of RGW01, RGW01A or RGW01B (SEQ IDNOS: 1, 2 or 3), or a variant thereof as defined in item I(ii) or (iii);and

b) at least one of the polypeptides of RGW03B, RGW03A or RGW03 (SEQ IDNOS: 9, 8 or 7), or a variant thereof as defined in item I(ii) or (iii);and

c) at least one of the polypeptides of RGW04 or RGW04A (SEQ ID NOS: 10or 11), or a variant thereof as defined in item I(ii) or (iii); andoptionally

d) at least one of the polypeptides of RGW02, RGW09, RGW06, RGW06A,RGW10 or RGW10A, RGW05 or RGW05A (SEQ ID NOS: 4, 26, 14, 15, 27, 12 or13), or a variant thereof as defined in item I(ii) or (iii); andoptionally

e) at least one of the polypeptides of RGW07, RGW07C or RGW07D (SEQ IDNOS: 16, 19 or 20), or a variant thereof as defined in item I(ii) or(iii).

XIII The composition according to any one of the preceding items,wherein one or more of the polypeptides have one or more modificationsselected from the following:

(i) N terminal acetylation;

(ii) C terminal amidation;

(iii) one or more hydrogen on the side chain amines of Arginine and/orLysine replaced with a methylene group;

(iv) glycosylation; and

(v) phosphorylation.

XIV. The composition according to any one of the preceding items whereinat least one of the peptides has been engineered to be soluble such thatit comprises:

i) N terminal to the residues of the peptide which flank a T cellepitope: one to six contiguous amino acids corresponding to the two tosix contiguous amino acids immediately N terminal to said residues inthe sequence of the protein from which the peptide derives; and/or

ii) C terminal to the residues of the peptide which flank a T cellepitope: one to six contiguous amino acids corresponding to the one tosix contiguous amino acids immediately C terminal to the said residuesin the sequence of the protein from which the peptide derives; or

iii) N and/or C terminal to the residues of the peptide which flank a Tcell epitope, at least one amino acid selected from arginine, lysine,histidine, glutamate and aspartate,

wherein the polypeptide has a solubility of at least 3.5mg/m1 and the Tcell epitope has a solubility of less than 3.5mg/ml.

XV. The composition according to any one of the preceding items whereinat least one of the peptides has been engineered to be soluble such thatadditionally:

i) any cysteine residues in the native sequence of the peptide arereplaced with serine or 2-aminobutyric acid; and/or

ii) any hydrophobic residues in the upto three amino acids at the Nand/or C terminus of the native sequence of the peptide, which are notcomprised in a T cell epitope, are deleted; and/or

iii) any two consecutive amino acids comprising the sequence Asp-Gly inthe upto four amino acids at the N and/or C terminus of the nativesequence of the peptide, which are not comprised in a T cell epitope,are deleted.

XVI. The composition according to any one of the preceding items whereineach polypeptide has a concentration in the range of 0.03 to 200nmol/ml, 0.3 to 200 nmol/ml or 30 to 120 nmol/ml.

XVII. A composition for use in preventing or treating allergy to ragweedby tolerisation comprising at least three polynucleotide sequences whichwhen expressed cause the production of a composition as defined in anyone of items Ito XVI.

XVIII. The composition according to item XVII, wherein eachpolynucleotide sequence capable of expressing a different polypeptide ispresent in the same or different polynucleotide vectors.

XIX. A vector for use in preventing or treating allergy to ragweed bytolerisation comprising at least three polynucleotide sequences whicheach encode a different polypeptide as defined in item I and optionallyone or more further polynucleotide sequences which encode differentpolypeptides as defined in item II.

XX. A vector for use in preventing or treating allergy to ragweed bytolerisation comprising between three and thirteen differentpolynucleotide sequences, which each encode a different polypeptide asdefined in item I or II, wherein at least one polynucleotide encodes apolypeptide selected from each of the following groups of polypeptides:

a) RGW01, RGW01A or RGW01B (SEQ ID NOS: 1, 2 or 3;

b) RGW03B, RGW03A or RGW03 (SEQ ID NOS: 9, 8 or 7); and

c) RGW04 or RGW04A (SEQ ID NOS: 10 or 11).

XXI. A product containing between three and thirteen polypeptides,wherein at least one polypeptide is selected from each of the followinggroups of polypeptides:

a) RGW01, RGW01A or RGW01B (SEQ ID NOS: 1, 2 or 3) or a variant thereofas defined in item I(ii) or (iii);

b) RGW03B, RGW03A or RGW03 (SEQ ID NOS: 9, 8 or 7) or a variant thereofas defined in item I(ii) or (iii); and

c) RGW04 or RGW04A (SEQ ID NOS: 10 or 11) or a variant thereof asdefined in item I(ii) or (iii);

wherein each different polypeptide is for simultaneous, separate orsequential use in preventing or treating allergy to ragweed bytolerisation.

XXII. A product containing between three and thirteen polynucleotidesequences, which each encode a different polypeptide as defined in itemI or II, wherein at least one polynucleotide encodes a polypeptideselected from each of the following groups of polypeptides:

a) RGW01, RGW01A or RGW01B (SEQ ID NOS: 1, 2 or 3;

b) RGW03B, RGW03A or RGW03 (SEQ ID NOS: 9, 8 or 7); and

c) RGW04 or RGW04A (SEQ ID NOS: 10 or 11);

and wherein each different polypeptide is for simultaneous, separate orsequential use in the prevention or treatment of allergy to ragweed in ahuman.

XXIII A pharmaceutical formulation for use in preventing or treatingallergy to ragweed by tolerisation comprising a composition according toany one of items I to XVIII; a vector according to any one of items XIXor XX; or a product according to any one of items XXI or XXII; and apharmaceutically acceptable carrier or diluent.

XXIV. The composition, vector or product according to item XXIII,formulated for oral administration, nasal administration, epicutaneousadministration, subcutaneous administration, sublingual administration,intradermal administration, buccal administration or for administrationby inhalation or by injection.

XXV. The composition as defined in any one of items Ito XVIII or productas defined in item XXI or XXII, additionally comprising a furtherpolypeptide allergen for use in tolerising an individual to the furtherpolypeptide allergen.

XXVI. An in vitro method of determining whether T cells recognize apolypeptide as defined in item I comprising contacting said T cells withsaid polypeptide and detecting whether said T cells are stimulated bysaid polypeptide.

XXVII. An in vitro method of determining whether an individual has or isat risk

of a condition wherein the condition is characterized by allergicsymptoms in

response to a ragweed allergen, the method comprising testing whetherthe individual has T cells which respond to a composition as defined inany one of items

I to XVIII, thereby determining whether the individual has or is at riskof the condition.

XXVIII. A method according to item XXVII wherein a T-cell immuneresponse to said composition is measured by contacting the compositionwith T cells in a sample taken from the subject, under conditions whichallow the composition and the T cells to interact; and determiningwhether or not any of the T cells are stimulated and thereby determiningwhether or not a T-cell immune response is present or absent.

1-15. (canceled)
 16. A composition suitable for use in preventing ortreating allergy to ragweed comprising at least three polypeptides,wherein the polypeptides are independently selected from the groupconsisting of polypeptides of any of SEQ ID NO's. 1 to 31 and variantsthereof.
 17. A composition according to claim 16, comprising at leastone polypeptide selected from the group consisting of GMIKSNDGPPI (SEQID NO: 1), GLIKSHDGPPV (SEQ ID NO: 2), GLIKSNDGPAA (SEQ ID NO: 3) andvariants thereof.
 18. A composition according to claim 17, wherein theat least one polypeptide is the polypeptide GMIKSNDGPPI (SEQ ID NO: 1),or a variant thereof.
 19. A composition according to claim 17,comprising at least one polypeptide selected from the group consistingof a polypeptide of RDLLENGAIFLPSG (SEQ ID NO: 9), DVFENGAIFVPSG (SEQ IDNO: 8), KDLLENGAIFVTSG (SEQ ID NO: 7) and variants thereof.
 20. Acomposition according to claim 19 wherein the at least one polypeptideis the polypeptide RDLLENGAIFLPSG (SEQ ID NO:9), or a variant thereof21. A composition according to claim 17 comprising at least onepolypeptide selected from selected from the group consisting of apolypeptide of KAGMIPAEPGEA (SEQ ID NO: 10), SAGMIPAEPGEA (SEQ ID NO:11), and variants thereof.
 22. A composition according to claim 17,comprising at least one polypeptide selected from the group consistingof a polypeptide of GSSQIWIDHSSLSKS (SEQ ID NO: 4), ETRRSLKTSGAYN (SEQID NO: 26), VVNSDKTIDGRGVKVE (SEQ ID NO: 14), AINNDKTIDGRGAKVE (SEQ IDNO: 15), FGFFQVVNNNYD (SEQ ID NO: 27), HGFFQVVNNNYD (SEQ ID NO: 28),KEGTLRFAAAQNRP (SEQ ID NO: 12), KEGTLRFGAAQNRP (SEQ ID NO: 13), andvariants thereof.
 23. A composition according to claim 17, comprising atleast one polypeptide selected from the group consisting of apolypeptide of GEAAIKLTSSAGVLS (SEQ ID NO: 16), KGEAAIKLTSSAGVLSK (SEQID NO: 19), KGEAAIKLTSSAGVLSKK (SEQ ID NO: 20) and variants thereof. 24.A composition according to claim 23, wherein the at least onepolypeptide is the polypeptide of KGEAAIKLTSSAGVLSKK (SEQ ID NO: 20) ora variant thereof.
 25. A composition according to claim 19 comprising atleast one polypeptide selected from selected from the group consistingof a polypeptide of KAGMIPAEPGEA (SEQ ID NO: 10), SAGMIPAEPGEA (SEQ IDNO: 11), and variants thereof. 26-28. (canceled)
 29. The compositionaccording to claim 16, wherein one or more of the polypeptides have oneor more modifications selected from the following: (i) N terminalacetylation; (ii) C terminal amidation; (iii) one or more hydrogen onthe side chain amines of Arginine and/or Lysine replaced with amethylene group; (iv) glycosylation; and (v) phosphorylation.
 30. Thecomposition according to claim 16, which is a solution in which eachpolypeptide has a concentration in the range of 0.03 to 200 nmol/ml. 31.The composition according to claim 16, which is a solution in which eachpolypeptide has a concentration in the range of 0.3 to 200 nmol/ml. 32.The composition according to claim 16, which is a solution in which eachpolypeptide has a concentration in the range of 30 to 200 nmol/ml. 33.The composition according to claim 16, which is a pharmaceuticalformulation comprising a pharmaceutically acceptable carrier or diluent.34. The pharmaceutical formulation according to claim 33, formulated fororal administration, nasal administration, epicutaneous administration,subcutaneous administration, sublingual administration, intradermaladministration, buccal administration or for administration byinhalation or by injection.
 35. A method of preventing or treatingallergy to ragweed, the method comprising administering to a subject inneed thereof a therapeutically effective amount of a compositioncomprising at least three polypeptides, wherein the polypeptides areindependently selected from the group consisting of polypeptides of anyof SEQ ID NO's. 1 to 31 and variants thereof.
 36. The method of claim35, wherein said subject is a human.
 37. The method of claim 35, whereinsaid subject is ragweed allergic.
 38. The method of claim 35, whereinsaid subject: is sensitised to Amb a 1; has a family history of allergyto ragweed; or displays symptoms of allergy when exposed to ragweed.